Hydroxy cyclohexenyl phenyl carboxamides tocolytic oxytocin receptor antagonists

ABSTRACT

The present invention provides substituted 10,11-Dihydro-5H-benzo[e]-pyrrolo[1,2-a][1,4]diazepine and 9,10-Dihydro-4H-3a,5,9-triaza-benzo[f]azulene compounds as well as methods and pharmaceutical compositions utilizing these compounds for the treatment and/or prevention and/or suppression of disorders which may be remedied or alleviated by oxytocin antagonist activity, including prevention and/or suppression of preterm labor, suppression labor at term prior to caesarean deliver, and for the treatment of dysmenorrhea. These compounds are also useful in enhancing fertility rates, enhancing survival rates and synchronizing estrus in farm animals.

This application claims priority from copending provisional applicationSer. No. 60/283,316, filed Apr. 12, 2001, the entire disclosure of whichis hereby incorporated by reference.

This invention concerns novel tricyclic hydroxy carboxamides which actas competitive oxytocin receptor antagonists, as well as methods oftheir manufacture, methods of treatment and pharmaceutical compositionsutilizing these compounds. The compounds of the present invention areuseful therapeutic agents in mammals, particularly in humans. Morespecifically, they can be used in the prevention and/or suppression ofpreterm labor, for the suppression of labor at term prior to caesareandelivery, to facilitate antinatal transport to a medical facility, andfor the treatment of dysmenorrhea. They are also useful in enhancingfertility rates, enhancing survival rates and synchronizing estrus infarm animals.

BACKGROUND OF THE INVENTION

Premature labor remains the leading cause of perinatal mortality andmorbidity. Infant mortality dramatically decreases with increasedgestational age. The survival rate of prematurely born infants increasesfrom 20% at 24 weeks to 94% at 30 weeks. Moreover the cost associatedwith the care of an infant born prematurely is extremely high. Whilemany agents have been developed for the treatment of premature labor inthe last 40 years, the incidence of pre-term births and low birth weightinfants has remained relatively unchanged. Therefore there remains anunmet need for the development of a safe and effective treatment ofpreterm labor.

Tocolytic (uterine relaxing) agents currently in use include β₂adrenergic receptor agonists such as Ritodrine which is moderatelyeffective in suppressing preterm labor, but it is associated withmaternal hypotension, tachycardia, and metabolic side effects. Severalother agents have been used to suppress premature labor, including otherβ₂ adrenergic agonists (terbutaline, albuterol), magnesium sulfate,NSAIDs (indomethacin), and calcium channel blockers. The consensus isthat none of these agents are very effective; there is no clinicalevidence showing that these compounds can prolong gestation for morethan 7 days (Johnson, Drugs, 45, 684-692 (1993)). Furthermore, theirsafety profile is not ideal. Adverse effects include respiratorydepression and cardiac arrest (magnesium sulfate), hemodynamic effects(calcium channel blockers), premature closure of the ductus arteriosusand oligohydramnios (NSAIDs; prostaglandin synthase inhibitors).Therefore, there is an unmet need for safer and more efficacious agentsfor the treatment of preterm labor with better patient tolerability.Specific requirements with regard to safety include a product with no orlow rates of tachycardia, limited anxiety, improved fetal safety, andfew, if any, adverse cardiovascular effects.

One target of interest is the oxytocin receptor in the uterus, and aselective oxytocin receptor antagonist has been proposed as an idealtocolytic agent. While the exact role of oxytocin (OT) in parturitionhas not been clearly defined, there is evidence strongly suggesting thatit may play a critical role in the initiation and progression of laborin humans (Fuchs et al. Science 215, 1396-1398 (1982); Maggi et al. J.Clin. Endocrinol. Metab. 70, 1142-1154 (1990); Akerlund, Reg. Pept 45,187-191 (1993); Akerlund, Int. Congr. Symp. Semin. Ser., Progress inEndocrinology 3, 657-660 (1993); Akerlund et al., in Oxytocin, Ed. R.Ivell and J. Russel, Plenum Press, New York, pp 595-600 (1995)).Preliminary clinical trials with oxytocin receptor antagonists supportthe concept that a blockade of OT receptors reduces uterine myometrialactivity and delays the onset of labor (Akerlund et al., Br. J. Obst.Gynaecol. 94, 1040-1044, (1987); Andersen et al., Am. J. Perinatol. 6,196-199 (1989); Melin, Reg. Pept 45, 285-288 (1993)). Thus, a selectiveoxytocin antagonist is expected to block the major effects of oxytocinexerted mainly on the uterus at term, and to be more efficacious thancurrent therapies for the treatment of preterm labor. By virtue of itsdirect action on the receptors in the uterus an oxytocin antagonist isalso expected have fewer side effects and an improved safety profile.

The following prior art references describe peptidic oxytocinantagonists: Hruby et al., Structure-Activity Relationships ofNeurohypophyseal Peptides, in The Peptides: Analysis, Synthesis andBiology; Udenfriend and Meienhofer Eds., Academic Press, New York, Vol.8, 77-207 (1987); Pettibone et al., Endocrinology, 125, 217 (1989);Manning et al., Synthesis and Some Uses of Receptor-Specific Agonistsand Antagonists of Vasopressin and Oxytocin, J. Recept Res., 13,195-214(1993); Goodwin et al., Dose Ranging Study of the Oxytocin AntagonistAtosiban in the Treatment of Preterm Labor, Obstet. Gynecol., 88,331-336 (1996). Peptidic oxytocin antagonists suffer from a lack of oralactivity and many of these peptides are non-selective antagonists sincethey also exhibit vasopressin antagonist activity. Bock et al. [J. Med.Chem. 33, 2321 (1990)], Pettibone et al. [J. Pharm. Exp. Ther. 256, 304(1991)], and Williams et al. [J. Med. Chem., 35, 3905 (1992)] havereported on potent hexapeptide oxytocin antagonists which also exhibitweak vasopressin antagonistic activity in binding to V₁ and V₂receptors.

Various non-peptidic oxytocin antagonists and/or oxytocin/vasopressin(AVP) antagonists have recently been reported by Pettibone et al.,Endocrinology, 125, 217 (1989); Yamamura et al., Science, 252, 572-574(1991); Evans et al., J. Med. Chem., 35, 3919-3927 (1992); Pettibone etal., J. Pharmacol. Exp. Ther, 264, 308-314 (1992); Ohnishi et al., J.Clin. Pharmacol. 33, 230-238, (1993); Evans et al., J. Med. Chem. 36,3993-4006 (1993); Pettibone et al., Drug Dev. Res. 30, 129-142 (1993);Freidinger et al., General Strategies in Peptidomimetic Design:Applications to Oxytocin Antagonists, in Perspect. Med. Chem. 179-193(1993), Ed. B. Testa, Verlag, Basel, Switzerland; Serradeil-LeGal, J.Clin. Invest., 92, 224-231 (1993); Williams et al., J. Med. Chem. 37,565-571 (1994); Williams et al., Bioorg. Med. Chem. 2, 971-985 (1994);Yamamura et al., Br. J. Pharmacol., 105, 546-551 (1995); Pettibone etal., Advances in Experimental Medicine and Biology 395, 601-612 (1995);Williams et al., J. Med. Chem. 38, 4634-4636 (1995); Hobbs et al.,Biorg. Med. Chem. Lett. 5, 1119 (1995); Williams et al., Curr. Pharm.Des. 2, 41-58 (1996); Freidinger et al., Medicinal Research Reviews, 17,1-16 (1997); Pettibone et al., Biochem. Soc. Trans. 25 (3), 1051-1057(1997); Bell et al., J. Med. Chem. 41, 2146-2163 (1998); Kuo et al.,Bioorg. Med. Chem. Lett. 8, 3081-3086 (1998); Williams et al., Biorg.Med. Chem. Lett. 9,1311-1316 (1999).

Certain carbostyril derivatives and bicyclic azepines are disclosed asoxytocin and vasopressin antagonists by Ogawa et al. in WO 94/01113(1994); benzoxazinones are disclosed as oxytocin and vasopressinreceptor antagonists by Sparks et al. in WO 97/25992 (1997); Williams etal. disclose piperidine oxytocin and vasopressin receptor antagonists inWO 96/22775 (1996); Bock et al. disclose benzoxazinone andbenzopyrimidinone piperidines useful as oxytocin and vasopressinreceptor antagonists in U.S. Pat. No. 5,665,719 (1997); piperazines andspiropiperidines useful as oxytocin and vasopressin receptor antagonistsare disclosed by Evans et al. in U.S. Pat. No. 5, 670,509 (1997) and byBock et al. in U.S. Pat. No. 5,756,504 (1998); Bell et al. disclosepiperazine oxytocin receptor antagonists in UK Patent Application, GB 2326 639 A (1998); Bell et al. disclose benzoxazinone and quinolinoneoxytocin and vasopressin receptor antagonists in UK Patent ApplicationGB 2 326 410 A (1998); Bell et al. disclose benzoxazinone oxytocin andvasopressin receptor antagonists in U.S. Pat. No. 5,756,497 (1998);Matsuhisa et al. disclose difluoro tetrahydrobenzazepine derivatives asoxytocin antagonists in WO 98/39325 (1998); and Ogawa et al. discloseheterocyclic bisamides with vasopressin and oxytocin antagonist activityin U.S. Pat. No. 5,753,644 (1998).

Trybulski et al. disclose 3-carboxamide derivatives ofpyrrolobenzodiazepine bisamides with vasopressin antagonist activity inU.S. Pat. No. 5,880,122 (1999); bicyclic thienoazepines with vasopressinand oxytocin receptor antagonist activity are disclosed by Albright etal. in WO 96/22294 (1996) and U.S. Pat. No. 5,654,297 (1997); andtricyclic benzazepines with vasopressin and oxytocin receptor antagonistactivity are disclosed by Albright et al. in U.S. Pat. No. 5,849,735(1998).

Albright et al. broadly disclose tricyclic benzazepine vasopressinantagonists in WO 96/22282A1 (1996).

Venkatesan et al. broadly disclose tricyclic benzazepines withvasopressin and oxytocin antagonist activity in U.S. Pat. No. 5,521,173(1996), WO 96/22292 (1996), and in U.S. Pat. No. 5,780,471 (1998).

Compounds which behave as potent oxytocin antagonists by binding withhigh affinity and selectivity to the oxytocin receptors, thus preventingoxytocin from binding to its receptors and exerting its biological andpharmacologic effects in vivo, can be useful for the treatment and/orprevention and/or suppression of preterm labor, for the suppression ofterm labor prior to a caesarian delivery, and to facilitate antinataltransport to a medical facility. They also can produce contraception inmammals given that oxytocin antagonists have been shown to inhibit therelease of oxytocin-stimulated luteneizing hormone (LH) from pituitarycells (Rettori et al., Proc. Nat. Acad. Sci. U.S.A. 94, 2741-2744(1997); Evans et al., J. Endocrinol., 122, 107-116 (1989); Robinson etal., J. Endocrinol. 125, 425-432 (1990)).

Oxytocin antagonists have the ability to relax uterine contractionsinduced by oxytocin in mammals and thus can be also useful for thetreatment of dysmenorrhea, a condition characterized by pain duringmenstruation (Åkerlund, Int. Congr. Symp. Semin. Ser., Progress inEndocrinology 3, 657-660 (1993); Åkerlund, Reg. Pept. 45, 187-191(1993); Melin, Reg. Pept. 45, 285-288 (1993)). Primary dysmenorrhea isassociated with ovulatory cycles, and it is the most common complaint ofgynecologic patients. Myometrial hypercontractility and decreased bloodflow to the uterus are thought to be causative factors for for thesymptoms of primary dysmenorrhea (Åkerlund, Acta Obstet. Gynecol. Scand.66, 459-461 (1987). In particular, vasoconstriction of small uterinearteries by vasopressin and oxytocin is thought to produce tissueischemia and pain (Jovanovic et al., Br. J. Pharmacol. 12, 1468-1474(91997); Chen et al., Eur. J. Pharmacol. 376, 25-51 (1999)).

The administration of oxytocin receptor antagonists to farm animalsafter fertilization has been found to enhance fertility rates byblocking oxytocin induced luteolysis leading to embryonic loss (Hickeyet al., WO 96/09824 A1 (1996), Sparks et al., WO 97/25992 A1 (1997);Sparks et al., U.S. Pat. No. 5,726,172 A (1998)). Thus, oxytocinreceptor antagonists can be useful in farm animal husbandry to controltiming of parturition and delivery of newborns resulting in enhancedsurvival rates. They can also be useful for the synchronization ofestrus by preventing oxytocin induced corpus luteum regression and bydelaying estrus (Okano, J. Reprod. Dev. 42 (Suppl.), 67-70 (1996)).Furthermore oxytocin receptor antagonists have been found to have apowerful effect in inhibiting oxytocin-induced milk ejection in dairycows (Wellnitz et al., Journal of Dairy Research 66, 1-8 (1999)).

Compounds which act as competitors of vasopressin binding to itsreceptors are useful in the treatment or prevention of state diseasesinvolving vasopressin disorders in mammals, which include vasodilationand aquaresis (free-water diuresis), treating hypertension andinhibiting platelet aggregation. They are useful in the treatment ofcongestive heart failure, cirrhosis with ascites, and in the syndrome ofinappropriate secretion of antiduretic hormone (SIADH). Furthermore,vasopressin receptor antagonists have been found to be useful intreating disturbances or illnesses of the inner ear, particularly thoserelated to Meniere's disease (Zenner et al., WO 99/2405-A2 (1999)); andfor the prevention and treatment of ocular circulatory disorders,particularly intraocular hypertension or glaucoma and vision disorderssuch as shortsightness (Ogawa et al., WO 99/38533-A1 (1999)).

SUMMARY OF THE INVENTION

This invention provides to novel compounds selected from those ofFormula (I):

R₁ and R₂ are, independently, selected from hydrogen, (C₁-C₆)loweralkyl, halogen, cyano, trifluoromethyl, hydroxy, amino, (C₁-C₆) loweralkylamino, (C₁-C₆) lower alkoxy, —OCF₃, (C₁-C₆) lower alkoxycarbonyl,—NHCO[(C₁-C₆)lower alkyl], carboxy, —CONH₂, —CONH(C₁-C₆) lower alkyl, or—CON[(C₁-C₆) lower alkyl]₂;

R₃ is a substituent selected from hydrogen, (C₁-C₆) lower alkyl, (C₁-C₆)lower alkoxy, hydroxy, amino, (C₁-C₆) lower alkylamino, —CO lower alkyl(C₁-C₆), or halogen;

R₄ consists of the moiety B-C;

wherein B is selected from the group of

and C is defined as:

wherein:

A is CH or N;

R₅, R₆, R₇ and R₈ are independently, selected from hydrogen, (C₁-C₆)lower alkyl, (C₁-C₆) lower alkoxy, (C₁-C₆) lower alkylcarbonyl, (C₃-C₆)lower alkenyl, (C₃-C₆) lower alkynyl, (C₁-C₆) lower alkyl, hydroxy(C₁-C₆) lower alkyl, alkoxy (C₁-C₆) lower alkyl, acyloxy (C₁-C₆),(C₃-C₈) cycloalkyl, formyl, cycloalkylcarbonyl, carboxy, alkoxycarbonyl,cycloalkyloxycarbonyl, aryl alkoxycarbonyl, carbamoyl, —O—CH₂—CH═CH₂,halogen, halo lower alkyl, trifluoromethyl, —OCF₃, —S(lower alkyl),—OC(O)N[lower alkyl]₂, —CONH(lower alkyl), —CON[lower alkyl]₂, loweralkylamino, di-lower alkylamino, lower alkyl di-lower alkylamino,hydroxy, cyano, trifluoromethylthio, nitro, amino, lower alkylsulfonyl,aminosulfonyl, lower alkylaminosulfonyl, phenyl, naphthyl, or

R₉ is chosen from the group of hydrogen, (C₁-C₆) lower alkyl,alkoxycarbonyl, —CON[(C₁-C₆) lower alkyl]₂, cyano; or aryl, optionallysubstituted by halogen, or (C₁-C₆) lower alkoxy;

R₁₀ represents one to two substituents chosen independently, from thegroup of hydrogen, (C₁-C₆) lower alkyl, [(C₁-C₆) lower alkyl]₂,carbonyl,

azido, amino, —NH[lower alkyl], —N[lower alkyl]₂, amino carbonyl loweralkyl, phthalimido, cyano, halogen, thio lower alkyl, aryloxy, arylthio,aryl optionally substituted with one to three substituents chosen from(C₁-C₆) lower alkyl, alkoxy or halogen;

hydroxy, lower alkoxy, —OSO₂R₃₂, or OP′ wherein P′ is tert-butyldimethylsilyl, tert-butyl diphenylsilyl, carbonyl loweralkyl, carbonyltrifluoro lower alkyl, aryl lower alkyl, arylcarbonyl, methoxymethyl, ormethylthiomethyl; with the proviso that when R₁₀ represents twosubstituents, the two substituents may be joined together to form withthe cyclohexene ring to which they are attached a bicyclic systemincluding but not limited to bicyclo[3.2.1]oct-2-ene, or(6,6-dimethyl)-bicyclo[3.1.1]hept-2-ene;

and R is selected from any of the following groups:

wherein:

R₁₁ is selected from the group of hydrogen, (C₁-C₆) lower alkyl,(C₇-C₁₂) aryl lower alkyl, wherein the aryl moiety is optionallysubstituted with lower alkoxy, or any of the following groups:

R₁₂ is selected from any of the group of:

R₁₃ and R₁₄ are independently, chosen from the group of hydrogen,(C₁-C₆) lower alkyl or (C₇-C₁₂) aryl lower alkyl;

R₁₅ is hydrogen, or (C₁-C₆) lower alkyl;

R₁₆ is hydroxy, (C₁-C₆) lower alkoxy, or OP wherein P is a hydroxyprotecting group, defined as a group providing temporary protectionagainst undesirable reactions during synthetic procedures and to beselectively removable. Common hydroxy protecting groups include, but arenot limited to, tert-butyidimethylsilyl, ter-butyldiphenylsilyl, acetyl,trifluoroacetyl, benzyl, benzoyl, methoxymethyl, methylthiomethyl, andothers well known in the art (c.f. Greene et al., Protective Groups inOrganic Syntheses, 3rd Edn., John Wiley & Sons, New York (1999));

R₁₇ is selected from the group of hydrogen, (C₁-C₆) lower alkyl or(C₇-C₁₂) aryl lower alkyl;

R₁₈ is selected from the group of —N[lower alkyl]₂, or —N[aryl loweralkyl]₂;

R₁₉ is hydrogen, or (C₁-C₆) lower alkyl, or R₂₇;

R₂₀ is selected from the group of (C₁-C₆) lower alkyl, —COR₁₆,—CONH[lower alkyl], —CON[lower alkyl]₂;

R₂₁ is aryl, optionally substituted by one to three substituents chosenfrom hydroxy, (C₁-C₆) lower alkoxy, aryloxy lower alkyl, or halogen;

R₂₂ represents one to four substituents chosen, independently, from thegroup of hydrogen or (C₁-C₆) lower alkyl;

R₂₃ is selected from the group of

R₂₄ is (C₁-C₆) lower alkyl, or aryl (C₁-C₆) lower alkyl;

R₂₅ and R₂₆ taken together represent one to four substituents chosen,independently, from the group of R₁₆, R₂₇, (C₁-C₆) lower alkyl, [(C₁-C₆)lower alkyl]₂, —CONH[lower alkyl], —CON[lower alkyl]₂, R₃₀,

with the proviso that at least one substituent is not (C₁-C₆) loweralkyl, —[(C₁-C₆) lower alkyl]₂, —CONH[(C₁-C₆) lower alkyl] or—CON[(C₁-C₆) lower alkyl]₂; and with further proviso that R₂₅ and R₂₆can be joined together to form a 5 or 6 membered saturated ringoptionally substituted by one or more substituents selected from R₁₆ orR₂₇;

R₂₇ and R₂₈ are selected from the group of hydroxy (C₁-C₆) lower alkyl,lower alkoxy (C₁-C₆) lower alkyl, or (C₁-C₆) lower alkyl OP, wherein Pis a hydroxy protecting group, defined as a group providing temporaryprotection against undesirable reactions during synthetic procedures andto be selectively removable. Common hydroxy protecting groups include,but are not limited to, tert-butyldimethylsilyl, ter-butyldiphenylsilyl,acetyl, trifluoroacetyl, benzyl, benzoyl, methoxymethyl,methylthiomethyl, and others well known in the art (c.f. Greene et al.,Protective Groups in Organic Syntheses, 3rd Edn., John Wiley & Sons, NewYork (1999));

R₂₉ represents one to four substituents chosen, independently, from thegroup of R₁₆ or R₂₇;

R₃₀ is

R₃₁ is hydrogen, or (C₁-C₆) lower alkyl;

X and Y are either CH, or N;

p is an integer from 0 to 1;

q is an integer from 2 to 4;

r is an integer from 0 to 3;

s is an integer from 0 to 2;

and t is an integer from 1 to 2;

and the pharmaceutically acceptable salts, or pro-drug forms thereof.

Among the more preferred compounds of this invention are those offormula:

R₁ and R₂ are, independently, selected from hydrogen, (C₁-C₆)loweralkyl, halogen, cyano, trifluoromethyl, hydroxy, amino, (C₁-C₆) loweralkylamino, (C₁-C₆) lower alkoxy, —OCF₃, (C₁-C₆) lower alkoxycarbonyl,—NHCO[(C₁-C₆)lower alkyl], carboxy, —CONH₂, —CONH(C₁-C₆) lower alkyl, or—CON[(C₁-C₆) lower alkyl]₂;

R₃ is a substituent selected from hydrogen, (C₁-C₆) lower alkyl, (C₁-C₆)lower alkoxy, hydroxy, amino, (C₁-C₆) lower alkylamino, —CO lower alkyl(C₁-C₆), or halogen;

R₄ consists of the moiety B-C;

wherein B is selected from the group of

and C is defined as:

wherein:

A is CH or N;

R₅, R₆, R₇ and R₈ are independently, selected from hydrogen, (C₁-C₆)lower alkyl, (C₁-C₆) lower alkoxy, (C₁-C₆) lower alkylcarbonyl, (C₃-C₆)lower alkenyl, (C₃-C₆) lower alkynyl, (C₁-C₆) lower alkyl, hydroxy(C₁-C₆) lower alkyl, alkoxy (C₁-C₆) lower alkyl, acyloxy (C₁-C₆),(C₃-C₈) cycloalkyl, formyl, cycloalkylcarbonyl, carboxy, alkoxycarbonyl,cycloalkyloxycarbonyl, aryl alkoxycarbonyl, carbamoyl, —O—CH₂—CH═CH₂,halogen, halo lower alkyl, trifluoromethyl, —OCF₃, —S(lower alkyl),—OC(O)N[lower alkyl]₂, —CONH(lower alkyl), —CON[lower alkyl]₂, loweralkylamino, di-lower alkylamino, lower alkyl di-lower alkylamino,hydroxy, cyano, trifluoromethylthio, nitro, amino, lower alkylsulfonyl,aminosulfonyl, or lower alkylaminosulfonyl;

R₉ is chosen from the group of hydrogen, (C₁-C₆)lower alkyl,alkoxycarbonyl, —CON[(C₁-C₆) lower alkyl]₂, or cyano;

R₁₀ represents one to two substituents chosen independently, from thegroup of hydrogen, (C₁-C₆) lower alkyl, [(C₁-C₆) lower alkyl]₂,carbonyl, azido, amino, —NH[lower alkyl], —N[lower alkyl]₂, aminocarbonyl lower alkyl, phthalimido, cyano, halogen, thio lower alkyl,aryloxy, arylthio, hydroxy, lower alkoxy, —OSO₂R₃₂, or OP′ wherein P′ istert-butyl dimethylsilyl, tert-butyl diphenylsilyl, carbonyl loweralkyl,carbonyl trifluoro lower alkyl, aryl lower alkyl, arylcarbonyl,methoxymethyl, or methylthiomethyl;

and R is selected from any of the following groups:

wherein:

R₁₁ is selected from the group of hydrogen, (C₁-C₆) lower alkyl,(C₇-C₁₂) aryl lower alkyl, wherein the aryl moiety is optionallysubstituted with lower alkoxy, or any of the following groups:

R₁₂ is selected from any of the following groups:

R₁₃ and R₁₄ are independently, chosen from the group of hydrogen,(C₁-C₆), lower alkyl or (C₇-C₁₂) aryl lower alkyl;

R₁₆ is hydroxy, (C₁-C₆) lower alkoxy, or OP wherein P is a hydroxyprotecting group, defined as a group providing temporary protectionagainst undesirable reactions during synthetic procedures and to beselectively removable. Common hydroxy protecting groups include, but arenot limited to, tert-butyidimethylsilyl, ter-butyldiphenylsilyl, acetyl,trifluoroacetyl, benzyl, benzoyl, methoxymethyl, methylthiomethyl, andothers well known in the art (c.f. Greene et al., Protective Groups inOrganic Syntheses, 3rd Edn., John Wiley & Sons, New York (1999));

R₁₇ is selected from the group of hydrogen, (C₁-C₆) lower alkyl or(C₇-C₁₂) aryl lower alkyl;

R₁₈ is selected from the group of —N[(C₁-C₆) lower alkyl]₂,

R₁₉ is hydrogen, or R₂₇;

R₂₀ is selected from the group of —COR₁₆, —CONH[(C₁-C₆) lower alkyl],—CON[(C₁-C₆) lower alkyl]₂;

R₂₁ is aryl, optionally substituted by one to three substituents chosenfrom hydroxy, or (C₁-C₆) lower alkoxy;

R₂₃ is selected from the group of

R₂₄ is (C₁-C₆) lower alkyl, or aryl (C₁-C₆) lower alkyl;

R₂₅ and R₂₆ taken together represent one to four substituents chosen,independently, from the group of R₁₆, R₂₇, (C₁-C₆) lower alkyl, [(C₁-C₆)lower alkyl]₂, —CONH[(C₁-C₆) lower alkyl], —CON[(C₁-C₆) lower alkyl]₂,R₃₀,

with the proviso that at least one substituent is not (C₁-C₆) loweralkyl, —[(C₁-C₆) lower alkyl]₂, —CONH[lower alkyl] or —CON[loweralkyl]₂; and with further proviso that R₂₅ and R₂₆ can be joinedtogether to form a 5 or 6 membered saturated ring optionally substitutedby one or more substituents selected from R₁₆ or R₂₇;

R₂₇ and R₂₈ are selected from the group of hydroxy (C₁-C₆) lower alkyl,lower alkoxy (C₁-C₆) lower alkyl, or (C₁-C₆) lower alkyl OP, wherein Pis a hydroxy protecting group, defined as a group providing temporaryprotection against undesirable reactions during synthetic procedures andto be selectively removable. Common hydroxy protecting groups include,but are not limited to, tert-butyldimethylsilyl, ter-butyidiphenylsilyl,acetyl, trifluoroacetyl, benzyl, benzoyl, methoxymethyl,methylthiomethyl, and others well known in the art (c.f. Greene et al.,Protective Groups in Organic Syntheses, 3rd Edn., John Wiley & Sons, NewYork (1999));

R₂₉ represents one to four substituents chosen, independently, from thegroup of R₁₆ or R₂₇;

R₃₀ is

R₃₁ is hydrogen, or (C₁-C₆) lower alkyl;

R₃₂ is selected from the group of (C₁-C₆) lower alkyl, trifluoro loweralkyl, or aryl optionally substituted by lower alkyl;

X and Y are either CH, or N;

p is an integer from 0 to 1;

q is an integer from 2 to 4;

r is an integer from 0 to 3;

s is an integer from 0 to 2;

and t is an integer from 1 to 2;

and the pharmaceutically acceptable salts, or pro-drug forms thereof.

The preferred compounds of this invention include:

-   1-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol;-   10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylic    acid;-   10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-(2,3-dihydroxypropyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   10-[4-(Cyclohex-1-en-1-yl)-3-methyl-benzoyl]-10,11-dohydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol;-   2,2,2-Trichloro-1-[10-(4-cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]-methanone;-   10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylic    acid;-   10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-((2S)-2-{(4R,5R)-5-[(1R)-1,2-dihydroxyethyl]-2-oxo-1,3-dioxolan-4-yl}-2-hydroxyethyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,    5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;-   (2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionic    acid ethyl ester;-   (2S)-2-{[10-(4-Cyclohex-1-en-1-yl)-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionic    acid;-   (2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionic    acid methyl ester;-   (2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionic    acid methyl ester;-   (2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionic    acid;-   (2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionic    acid;-   10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-depxy-D-glucitol;-   10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylic    acid;-   {10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}-3-carboxylic    acid bis-(2-hydroxy-ethyl)-amide; and-   {10-[4-(1-Cyclohexen-1-yl)-3-methylbenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}[4-(2-hydroxyethyl)-1-piperazinyl]methanone;    or a pharmaceutically acceptable salt form thereof.

It is understood by those practicing the art that some of the compoundsof this invention depending on the definition of R₁, R₂, R₃, R₄, and Rmay contain one or more asymmetric centers and may thus give rise toenantiomers and diastereomers. The present invention includes allstereoisomers including individual diastereomers and resolved,enantiomerically pure R and S stereoisomers; as well as racemates, andall other mixtures of the R and S stereoisomers and pharmaceuticallyacceptable salts thereof, which possess the indicated activity. Opticalisomers may be obtained in pure form by standard procedures known tothose skilled in the art. It is also understood that this inventionencompasses all possible regioisomers, E-Z isomers, endo-exo isomers,and mixtures thereof which possess the indicated activity. Such isomersmay be obtained in pure form by standard separation procedures known tothose skilled in the art. It is understood also by those practicing theart that some of the compounds of this invention depending on thedefinition of R₅, R₆, R₇, R₉ and R₁₀ may be chiral due to hinderedrotation, and give rise to atropisomers which can be resolved andobtained in pure form by standard procedures known to those skilled inthe art. Also included in the present invention are all polymorphs andhydrates of the compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises the compounds described above, as wellas pharmaceutical compositions containing the compounds of thisinvention in combination or association with one or morepharmaceutically acceptable carrier or excipient. In particular, thepresent invention provides a pharmaceutical composition which comprisesa therapeutically effective amount of one or more compounds of thisinvention in a pharmaceutically acceptable carrier or excipient.

This invention also comprises methods for treating conditions in amammal, preferably a human, which are remedied or alleviated by oxytocinantagonist activity including, but not limited to, treatment orprevention of preterm labor, dysmenorrhea and suppressing labor prior tocaesarian delivery whenever desirable in a mammal, preferably in ahuman. The methods comprise administering to a mammal in need thereof atherapeutically effective but non-toxic amount of one or more of thecompounds of this invention.

The present invention also comprises combinations of the compounds ofthe present invention with one or more agents useful in the treatment ofdisorders such as preterm labor, dysmenorrhea, and stopping labor priorto caesarian delivery. More specifically, the compounds of the presentinvention may be effectively administered in combination with effectiveamounts of other tocolytic agents used in the treatment or prevention ofpreterm labor, dysmenorrhea or suppressing labor prior to caesareandelivery including β-adrenergic agonists, calcium channel blockers,prostaglandin synthesis inhibitors, other oxytocin antagonists (e.g.atosiban), magnesium sulfate, ethanol, and other agents useful in thetreatment of said disorders. The present invention is to be understoodas embracing all simultaneous or alternating treatments of anycombination of the compounds of the present invention with othertocolytic agents with any pharmaceutical composition useful for thetreatment of preterm labor, dysmenorrhea, and suppressing labor prior tocaesarean delivery in mammals.

The compositions are preferably adapted for intravenous (both bolus andinfusion) and oral administration. However, they may be adapted forother modes of administration including subcutaneous, intraperitoneal,or intramuscular administration to a human or a farm animal in need of atocolytic agent.

The compounds of the present invention can be used in the form of saltsderived from non toxic pharmaceutically acceptable acids or bases. Thesesalts include, but are not limited to, the following: salts withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and, as the case may be, such organicacids as acetic acid, oxalic acid, citric acid, tartaric acid, succinicacid, maleic acic, benzoic acid, benzene sulfonic acid, fumaric acid,malic acid, methane sulfonic acid, pamoic acid, and para-toluenesulfonic acid. Other salts include salts with alkali metals or alkalineearth metals, such as sodium, potassium, calcium or magnesium, or withorganic bases including quaternary ammonium salts. The compounds canalso be used in the form of esters, carbamates and other conventionalprodrug forms, which in general, will be functional derivatives of thecompounds of this invention which are readily converted to the activemoiety in vivo. This is meant to include the treatment of the variousconditions described hereinbefore with a compound of this invention orwith a compound which is not specifically disclosed but which convertsto a compound of this invention in vivo upon administration. Alsoincluded are metabolites of the compounds of the present inventiondefined as active species produced upon introduction of these compoundsinto a biological system.

When the compounds of this invention are employed for the aboveutilities, they may be combined with one or more pharmaceuticallyacceptable excipients or carriers, for example, solvents, diluents andthe like, and may be administered orally in such forms as tablets,capsules (including time release and sustained release formulations),pills, dispersible powders, granules, or suspensions containing, forexample, from 0.05 to 5% of suspending agent, syrups containing, forexample, from about 10 to 50% of sugar, and elixirs and the like, orparenterally in the form of sterile injectable solutions, suspensions oremulsions containing from about 0.05 to 5% suspending agent in anisotonic medium. Such pharmaceutical preparations may contain, forexample, from about 25 to about 90% of the active ingredient incombination with the carrier, more usually between about 5% and 60% byweight.

The effective dosage of active ingredients employed may vary dependingon the particular compound or salt employed, the mode of administration,age, weight, sex and medical condition of the patient, and the severityof the condition being treated. An ordinarily skilled physician,veterinarian or clinician can readily determine and prescribe theeffective amount of the agent required to prevent, counter or arrest theprogress of the condition. However, in general, satisfactory results areobtained when the compounds of the invention are administered at a dailydose of from about 0.5 to about 500 mg/Kg of mammal body weight,preferably given in divided doses two to four times a day, or in asustained release form. For most large mammals the total daily dosage isfrom about 0.5 to 100 mg, preferably from 0.5 to 80 mg/Kg. Dosage formssuitable for internal use comprise from about 0.05 to 500 mg of theactive compound in intimate admixture with a solid or liquidpharmaceutically acceptable carrier. This dosage regimen may be adjustedto provide the optimal therapeutic response. For example, severaldivided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

These active compounds may be administered orally as well as byintravenous, intramuscular, or subcutaneous routes. Solid carriersinclude starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, glycerol, non-ionic surfactants and edibleoils such as corn, peanut and sesame oils, as are appropriate to thenature of the active ingredient and the particular form ofadministration desired. Adjuvants customarily employed in thepreparation of pharmaceutical compositions may be advantageouslyincluded, such as flavoring agents, coloring agents, preserving agents,and antioxidants, for example vitamin E, ascorbic acid, BHT and BHA.

These active compounds may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy injectability exists. It must be stable underconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol (e.g. glycerol, propylene glycol, and liquid polyethyleneglycol), suitable mixtures thereof, and vegetable oil.

Furthermore, active compounds of the present invention can beadministered intranasally using vehicles suitable for intranasaldelivery, or transdermally using transdermal skin patches known to thoseordinarily skilled in the art. When using a transdermal delivery system,the dosage administration will be continuous rather than in a single ordivided daily doses. The compounds of the present invention can also beadministered in the form of liposome delivery system wherein theliposomal lipid bilayers are formed from a variety of phospholipids.

Compounds of the present invention may also be delivered by the use ofcarriers such as monoclonal antibodies to which the active compounds arecoupled. The compounds of the present invention may also be coupled tosoluble polymers as drug carriers or to biodegradable polymers useful inachieving controlled release of the active agent.

Also according to the present invention there are provided processes forproducing the compounds of the present invention.

Process of the Invention

The compounds of the present invention may be prepared according to oneof the general processes outlined below.

The compounds of general formula (I) wherein

R, R₃, and R₄ are defined hereinbefore, can be conveniently prepared asshown in Scheme I.

According to the above preferred process, a tricyclic diazepine offormula (1) wherein

R₃ and R₄ are defined hereinbefore, is reacted with perhaloalkanoylhalide preferably trichloroacetyl chloride in the presence of an organicbase such as N,N-diisopropyl ethyl amine (Hünig's base) in an aproticorganic solvent such as dichloromethane at temperatures ranging from−10° C. to ambient to provide the desired trichloroacetyl intermediateof formula (2). Subsequent hydrolysis of (2) with aqueous base such assodium hydroxide in an organic solvent such as tetrahydrofuran oracetone at temperatures ranging from −10° C. to ambient, yields theintermediate acid of formula (3). The required activation of thecarboxylic acid (3) for the subsequent coupling with a primary orsecondary amine of formula (5) can be accomplished in several ways.Thus, (3) can be converted to an acid halide preferably a chloride orbromide of formula (4, J=COCl or COBr) by reaction with thionylchloride(bromide) or oxalyl chloride(bromide) or similar reagents knownin the art, either neat or in the presence of an inorganic base such aspotassium carbonate, or in the presence of an organic base such aspyridine, 4-(dimethylamino)pyridine, or a tertiary amine such astriethylamine in an aprotic solvent such as dichloromethane,N,N-dimethylformamide or tetrahydrofuran at temperatures ranging from−5° C. to 50° C. to yield the intermediate acylated derivative (4).Subsequent coupling of the acid chloride(bromide) (4, J=COCl or COBr)with an appropriately substituted primary or secondary amine of formula(5) in the presence of a stoichiometric amount of Hünig's base in anaprotic solvent such as dichloromethane, N,N-dimethylformamide ortetrahydrofuran at temperatures ranging from ambient to the refluxtemperature of the solvent provides the desired compounds of formula (I)wherein

R, R₃ and R₄ are as defined hereinbefore.

Alternatively, the acylating species can be a mixed anhydride of thecorresponding carboxylic acid, such as that prepared by treating saidacid of formula (3) with 2,4,6-trichlorobenzoyl chloride in an aproticorganic solvent such as dichloromethane according to the procedure ofInanaga et al., Bull. Chem. Soc. Jpn. 52, 1989 (1979). Treatment of saidmixed anhydride of formula (4) with an appropriately substituted primaryor secondary amine of formula (5) in an aprotic solvent such asdichloromethane at temperatures ranging from ambient to the refluxtemperature of the solvent provides the desired compounds of formula (I)wherein

R, R₃ and R₄ are as defined hereinbefore.

Alternatively, amidation of the carboxylic acids of formula (3) can beeffectively carried out by treatment of said acid with triphosgene in anaprotic solvent such as dichloromethane followed by reaction of theactivated intermediate with an appropriately substituted primary orsecondary amine of formula (5) in the presence of an organic base suchas Hünig's base at temperatures ranging from −10° C. to ambient.

Another preferred process for the preparation of the compounds of thepresent invention of formula (I) wherein

R, R₃ and R₄ are as defined hereinbefore, consists of treating the acidof formula (3) with an activating reagent such asN,N-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride in the presence of 1-hydroxybenzotriazolefollowed by reaction of the activated intermediate with an appropriatelysubstituted primary or secondary amine of formula (5) preferably in thepresence of an organic base such as Hünig's base and a catalytic amountof 4-(dimethylamino)pyridine in an aprotic solvent such asdichloromethane, N,N-dimethylformamide or tetrahydrofuran attemperatures ranging from −10° C. to ambient.

In another preferred process, said acid (3) can be activated bytreatment with other activating agents such as N,N′-carbonyldiimidazolein an aprotic solvent such as dichloromethane or tetrahydrofuran attemperatures ranging from −10° C. to the reflux temperature of thesolvent. Subsequent reaction of the intermediate activated imidazolidewith an appropriately substituted primary or secondary amine of formula(5) provides the desired compounds of formula (I) wherein

R, R₃ and R₄ are as defined hereinbefore.

Alternatively, the coupling of the appropriately substituted primary orsecondary amine of formula (5) with said acid of formula (3) can beeffectively carried out by using hydroxybenzotriazole tetramethyluroniumhexafluorophosphate as the coupling reagent in the presence of anorganic base such as Hünig's base and in a solvent such asN,N-dimethylformamide at temperatures ranging from −10° C. to ambient toprovide in good isolated yield and purity the desired compounds offormula (I) wherein

R, R₃ and R₄ are as defined hereinbefore.

Related coupling reagents such as diphenylphosphoryl azide, diethylcyano phosphonate, benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate and all other known in the literaturethat have been used in the formation of amide bonds in peptide synthesiscan also be used for the preparation of compounds of formula (I) wherein

R, R₃ and R₄ are as defined hereinbefore.

As an alternative, reaction of the intermediate 3-trihalomethylketone offormula (2) directly with an appropriately substituted primary orsecondary amine of formula (5) also provides the desired compounds offormula (I) wherein

R, R₃ and R₄ are as defined hereinbefore.

The method of choice for the preparation of compounds of formula (I)from the intermediate carboxylic acid (3) is ultimately chosen on thebasis of its compatibility with the R, R₃ and R₄ groups, and itsreactivity with the tricyclic benzodiazepine of formula (I).

Another preferred process for the preparation of (I) of Scheme I isshown in Scheme II. A tricyclic diazepine of formula (1) is reacted withdiphosgene in an aprotic solvent such as dichloromethane preferably inthe presence of an organic base such as triethylamine, followed byreaction of the resulting acylated intermediate with an appropriatelysubstituted primary or secondary amine of formula (5) to provide thedesired compounds of formula (I) wherein

R, R₃ and R₄ are as defined herein before.

The tricyclic diazepines of formula (1) of Scheme (I) wherein R₄ isdefined hereinbefore, can be conveniently prepared as shown in SchemeIII.

Thus, a tricyclic diazepine of formula (6) is treated with anappropriately substituted acylating agent such as a haloaroyl halide,preferably an appropriately substituted acyl chloride or bromide offormula (7, J=COCl or COBr) wherein R₄ is ultimately chosen on the basisof its compatibility with the present reaction scheme, in the presenceof an inorganic base such as potassium carbonate, or in the presence ofan organic base such as pyridine, 4-(dimethylamino)pyridine, or atertiary amine such as triethylamine or N,N-diisopropylethylamine in anaprotic solvent such as dichloromethane, N,N-dimethylformamide ortetrahydrofuran, at temperatures ranging from −5° C. to 50° C. toprovide intermediates of general formula (1) wherein R₄ is definedhereinbefore.

Alternatively, the acylating species of formula (7) can be a mixedanhydride of the corresponding carboxylic acid, such as that prepared bytreating said acid with 2,4,6-trichlorobenzoyl chloride in an aproticorganic solvent such as dichloromethane according to the procedure ofInanaga et al., Bull. Chem. Soc. Jpn., 52, 1989 (1979). Treatment ofsaid mixed anhydride of general formula (7) with a tricyclic diazepineof formula (6) in a solvent such as dichloromethane, and in the presenceof an organic base such as 4-(dimethylamino)pyridine, at temperaturesranging from 0° C. to the reflux temperature of the solvent, yields theintermediate acylated derivative (1) of Scheme III.

The acylating intermediate of formula (7) is ultimately chosen on thebasis of its compatibility with the R₄ groups, and its reactivity withthe tricyclic diazepine of formula (6).

The desired intermediates of formula (7) of Scheme III wherein R₄consists of the moiety B-C wherein B is (a) and C is (c) can beconveniently prepared by a process shown in Scheme IV. Thus, anappropriately substituted aryl(heteroaryl) iodide(bromide) of formula(8) wherein P is a carboxylic acid protecting group, preferably P=alkylor benzyl, M=I, and A, R₅, R₆ and R₇ are defined hereinbefore, isreacted with a tri(alkyl)tin(IV) derivative of formula (9,W=Sn(trialkyl)₃, preferably Sn(n-Bu)₃) wherein R₉, R₁₀ are ultimatelychosen on the basis of their compatibility with the present reactionscheme, in the presence of a Pd(0) catalyst, and in the presence orabsence of inorganic salts (e.g. LiCl), in an aprotic solvent such asdioxane or N-methylpyrrolidinone, to provide the intermediate ester(10). Subsequent unmasking of the carboxylic acid by hydrolysis,hydrogenolysis or similar methods known in the art, followed byactivation of the intermediate acid (11) provide the desired compoundsof formula (19) wherein A, R₅, R₆, R₇, R₈, R₉ and R₁₀ are hereinbeforedefined, suitable for coupling with the tricyclic diazepine of formula(6).

The desired intermediates of formula (7) of Scheme III wherein R₄consists of the moiety B-C where B is (a) and C is (d) or B is (b) and Cis either (c) or (d), can be prepared by a process analogous to thatexemplified in Scheme IV by replacing intermediates of formulas (8 and9) with appropriately substituted naphthyl, dihyfronaphthyl ordihydroquinolinyl intermediates.

Alternatively, the desired intermediates of formula (10) of Scheme IVwherein R₄ consists of the moiety B-C where B is (a) and C is (c) can beprepared by Suzuki coupling from the iodide(bromide,trifluoromethanesulfonate) (8, M=I, Br or OTf) and an appropriatelysubstituted boron derivative of formula (9, preferably W=B(OH)₂) in thepresence of a palladium catalyst such as palladium(II) acetate ortetrakis(triphenylphosphine) palladium(0) and an organic base such astriethylamine or an inorganic base such as sodium(potassium or cesium)carbonate with or without added tetrabutylammonium bromide(iodide), in amixture of solvents such as toluene-ethanol-water, acetone-water, wateror water-acetonitrile at temperatures ranging from ambient to the refluxtemperature of the solvent (Suzuki, Pure & Appl. Chem. 66, 213-222(1994), Badone et al., J. Org. Chem. 62, 7170-7173 (1997)). The exactconditions for the Suzuki coupling of the halide and the boronic acidintermediates are chosen on the basis of the nature of the substrate andthe substituents. The desired intermediates of formula (10) of Scheme IVcan be similarly prepared from the bromide (8, M=Br) and the boronicacid (9) in a solvent such as dioxane, N,N-dimethylformamide ordimethylsulfoxide, in the presence of potassium phosphate and a Pd(0)catalyst.

Alternatively, a cross coupling reaction of an iodide (bromide ortrifluoromethanesulfonate) of formula (9, W=Br, I, OTf) with abis(pinacolato)diboron [boronic acid, or trialkyl tin(IV)] derivative offormula (8,

B(OH)₂, or SnBu₃) yields the desired intermediate of formula (10) whichis converted to (I) in the manner of Scheme IV.

The desired intermediates of formula (10) of Scheme IV wherein R₄consists of the moiety B-C wherein B is (a) and C is (d) or B is (b) andC is either (c) or (d) can be prepared in analogous fashion by replacingintermediates of formulas (8 and 9) with appropriately substitutednaphthyl, dihydronaphthyl, or dihydroquinolyl intermediates.

The required appropriately substituted aryl(heteroaryl) halides offormula (8, M=Br or I) of Scheme IV are either available commercially,or are known in the art or can be readily accessed in quantitativeyields and high purity by diazotization of the corresponding substitutedanilines (8, P=H, alkyl or benzyl, M=NH₂) followed by reaction of theintermediate diazonium salt with iodine and potassium iodide in aqueousacidic medium essentially according to the procedures of Street et al,J. Med. Chem. 36, 1529 (1993) and Coffen et al., J. Org. Chem. 49, 296(1984) or with copper(I) bromide, respectively (March, Advanced OrganicChemistry, 3^(rd) Edn., p.647-648, John Wiley & Sons, New York (1985).

Alternatively, the desired intermediates of formula (11, A=CH) of SchemeIV wherein R₄ consists of the moiety B-C wherein B is (a), A=CH) and Cis (c) can be conveniently prepared as shown in Scheme V bycross-coupling reaction of an appropriately substituted pinacolatoborane of formula (13) wherein R₉, R₁₀ are ultimately chosen on thebasis of their compatibility with the present reaction scheme, with anaryl triflate of formula (14, Y=OTf) or an aryl halide (14, Y=Br, I)wherein R₅, R₆ and R₇ are defined hereinbefore, according to the generalprocedures of lshiyama et al., Tetr. Lett. 38, 3447-3450 (1997) andGiroux et al. Tetr. Lett. 38, 3841-3844 (1997), followed by basic oracidic hydrolysis of the intermediate nitrile of formula (15) (cf.March, Advanced Organic Chemistry, 3^(rd) Edn., John Wiley & Sons, NewYork, p. 788 (1985)).

Alternatively, reaction of an iodide (bromide, ortrifluoromethanesulfonate) of formula (12, X=Br, I, or OTf) with abis(pinacolato)diboron [boronic acid or trialkyl tin(IV)] derivative offormula (14,

B(OH)₂, or SnBu₃) yields the desired intermediate of formula (15) whichis converted to (6) in the manner of Scheme V.

The desired intermediates of formula (11) of Scheme IV wherein R₄consists of the moiety B-C wherein B is (a, A=CH) and C is (d) or B is(b) and C is either (c) or (d, A=CH), can be prepared in analogousfashion by replacing intermediates of formulas (13 and 14) withappropriately substituted naphthyl or dihydronaphthyl intermediates.

The desired boronic esters of formula (13) of Scheme V can beconveniently prepared by the palladium-catalyzed cross-coupling reactionof the pinacol ester of diboronic acid (16) with an appropriatelysubstituted alkenyl halide preferably a bromide or iodide (12, X=Br, I)or alkenyl triflate (12, X=OTf) according to the described procedures oflshiyama et al., J. Org. Chem. 60, 7508-7510 (1995) and Giroux et al.,Tetr. Lett. 38, 3841-3844 (1997).

The desired compounds of formula (1) of Scheme IV wherein R₄ consists ofthe moiety B-C wherein B is (a) and C is (c) can be alternativelyprepared by a process shown in Scheme VI.

Thus, a tricyclic diazepine of formula (6) is treated with anappropriately substituted acylating agent such as a haloaroyl(heteroaroyl)halide, preferably an iodo(bromo) aroyl(heteroaroyl)chloride(bromide) of formula (17, J=COCl or COBr; X=I, Br) wherein R₅,R₆ and R₇ are hereinbefore defined using any of the procedureshereinbefore described, to provide the acylated intermediate of generalformula (18) of Scheme VI.

Alternatively, the acylating species of formula (17) can be a mixedanhydride of the corresponding carboxylic acid. Treatment of said mixedanhydride of general formula (17) with a tricyclic diazepine of formula(6) according to the procedure described hereinbefore yields theintermediate acylated derivative (18).

The acylating intermediate of formula (17) is ultimately chosen on thebasis of its compatibility with A and the R₅, R₆ and R₇ groups, and itsreactivity with the tricyclic benzodiazepine of formula (6).

A Stille coupling reaction of (18, X=I) with an appropriatelysubstituted organotin reagent such as a trialkyltin(IV) derivative,preferably a tri-n-butyltin(IV) derivative of formula (9, W=SnBu₃)wherein R₉ and R₁₀ are ultimately chosen on the basis of theircompatibility with the present reaction scheme, in the presence of acatalyst such as tetrakis (triphenylphosphine) palladium (0), in anaprotic organic solvent such as toluene or N,N-dimethylformamide, attemperatures ranging from ambient to 150° C. (cf. Farina et al., J. Org.Chem, 59, 5905 (1994) and references cited therein) affords the desiredcompounds of formula (1) wherein

A, R₃, R₅, R₆, R₇, R₇, R₉ and R₁₀ are as defined hereinbefore.

Alternatively, reaction of a compound of formula (18, X=Cl, Br or I)with an appropriately substituted boronic acid of formula (9, W=B(OH)₂),wherein R₉ and R₁₀ are chosen on the basis of their compatibility withthe reaction scheme, in a mixture of solvents such astoluene-ethanol-water, and in the presence of a Pd(0) catalyst and abase such as sodium carbonate, at temperatures ranging from ambient tothe reflux temperature of the solvent, yields the desired compounds offormula (1) wherein

A, R₃, R₅, R₆, R₇, R₈, R₉ and R₉ are as defined hereinbefore.

Alternatively, a cross-coupling reaction of a compound of formula (18,X=Br or I) with a bis(pinacolato) diboron of formula (16) in thepresence of a catalyst such asdichloro-[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct and potassium acetate, in an aprotic solvent suchas dimethylsulfoxide, at temperatures ranging from ambient to 100° C.,yields the intermediate of formula (18,

Subsequent reaction of (18) with an appropriately substitutedtrifluoromethane sulfonate of formula (9, W=OTf) in the presence of abase such as aqueous sodium carbonate, in an aprotic solvent such asN,N-dimethylformamide, at temperatures ranging from ambient to thereflux temperature of the solvent, provides the desired compounds offormula (1) wherein

A, R₃, R₅, R6, R₇, R₈, R₉ and R₉ are as defined hereinbefore.

The preferred substituted aroyl(heteroaroyl) chlorides(bromides) offormula (17) of Scheme VI(X=I, Br; J=COCl or COBr) wherein A, R₅, R₆ andR₇ are as defined hereinbefore, are either available commercially, orare known in the art, or can be readily prepared by procedures analogousto those in the literature for the known compounds.

The intermediates of formula (9, W=Sn(alkyl)₃, alkyl=n-butyl) of SchemeVI are either commercially available or can be conveniently prepared asshown in Scheme VII from the corresponding bromo starting materials offormula (20) wherein R₉, R₁₀ are ultimately chosen on the basis of theircompatibility with the reaction scheme, by first reacting them withn-butyl lithium followed by reaction of the intermediate lithiatedspecies with a trialkyl (preferably trimethyl or tri-n-butyl)tin(IV)chloride).

The preferred substituted boronic acids of formula (9, W=B(OH)₂) areeither available commercially, or are known in the art, or can bereadily prepared by procedures analogous to those in the literature forthe known compounds.

The desired compounds of formula (1) of Scheme VI wherein R₄ consists ofthe moiety B-C wherein B is (a) and C is (d) or B is (b) and C is either(c) or (d) can be prepared in analogous fashion by replacingintermediates of formulas (17 and 9) with appropriately substitutednaphthyl, dihydronaphthyl or dihydroquinolinyl intermediates.

Alternatively, as shown in Scheme VIII, the appropriately substitutedaroyl(heteroaroyl) halides, preferably aroyl(heteroaroyl) chlorides offormula (21, J=COCl) where A, R₅, R₆ and R₇ are hereinbefore defined,are reacted with a tricyclic diazepine of formula (6) to provide theintermediate bromides of formula (22). Subsequent reaction of (22) witha bis-alkyl-tin reagent (preferably bis-(tri-n-butyl)-tin(IV)) in thepresence of a Pd(0) catalyst such astetrakis(tri-phenylphosphine)palladium(0) and lithium chloride, providesthe stannane intermediate of formula (23). Further reaction of thetri-n-butyl tin(IV) derivative (23) with the appropriately substitutedalkenyl halide of formula (24, M =bromo or iodo) wherein R₉, R₁₀ areultimately chosen on the basis of their compatibility with the presentreaction scheme, in the presence of a Pd(0) catalyst such astetrakis(triphenylphosphine) palladium(0), yields the desired compoundsof formula (1) wherein R4 consists of the moiety B-C wherein B is (a)and C is (c), and

A, R₅, R₆, R₇, R₉ and R₁₀ are defined hereinbefore.

The desired compounds of formula (1) of Scheme VIII wherein R₄ consistsof the moiety B-C wherein B is (a) C is (d) and B is (b) and C is either(c) or (d) can be prepared in analogous fashion by replacingintermediates of formulas (21 and 24) with appropriately substitutednaphthyl, dihydronaphthyl or dihydroquinolinyl intermediates.

A preferred process for the preparation of the compounds of formula (1)of Scheme I wherein

R₃, R₅, R₆ and R₇ are defined hereinbefore, and R4 consists of themoiety B-C wherein B is (a, A=CH) and C is (c), and R₁₀ is hydroxy,alkoxy, OP′, azido, phthalimido, cyano, phenoxy, thiophenoxy, thioalkyl,and related nucleophiles, is shown in Scheme IX.

According to the preferred process, an appropriately substituteddiazepine cyclohexenone of formula (25) is converted to thecorresponding cyclohexenol (26) by reduction with a metal hydridepreferably sodium borohydride in the presence of cerium (III) chloride,in an hydroxylic solvent such as methanol, at temperatures ranging from−78° C. to ambient. The hydroxy function of (26) is then activated byconversion to a leaving group (28, L=leaving group) preferably apara-toluenesulfonate, methanesulfonate, trifluoromethanesulfonate,phosphate and the like. S_(N)2 displacement of the leaving group with anucleophile such as azide, phthalimide, cyamide, halide, phenol, carbonor sulfur nucleophiles and the like, provides the desired compound (1)wherein

and R₃ are defined hereinbefore, R₄ consists of the moiety B-C wherein Bis (a, A=CH) and C is (c), and R₅, R₆, R₇, R₉ and R₁₀ are definedhereinbefore.

Alternatively, the chiral cyclohexenol of formula (26) is separated bychiral HPLC into its respective enantiomers of formula (28) and (29)according to Scheme X. Each enantiomer can be individually activated andsubjected to S_(N)2 displacement with a nucleophile in the manner ofScheme IX.

Alternatively, the chiral cyclohexenols of formula (28) and (29) areobtained by asymmetric reduction of the cyclohexenone of formula (25)with a borane-tetrahydrofuran complex in an aprotic solvent such astetrahydrofuran in the presence of a chiral auxiliary such as(S)-tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaboroleor(R)-tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaborolerespectively, at ambient temperature.

Preferred processes for the preparation of compounds of formula (I) ofScheme I wherein R₄ consists of the moiety B-C wherein B is (a) and C is(d) or B is either (b) or (c) and

A, R, R₃, R₅, R₆, R₇ are defined hereinbefore, and wherein R₉ and R₁₀are ultimately chosen on the basis of their compatibility with thereaction scheme, also utilize acylation of the amide intermediate (31)with an acylating agent of formula (17) of Scheme VI, as shown in SchemeXI.

Alternatively, the preferred compounds of formula (I) of scheme Iwherein R₄ consists of the moiety B-C wherein B is (a) and C is (c) and

A, R, R₃, R₅, R₆, R₇ are defined hereinbefore, and wherein R₉, R₁₀ areultimately chosen on the basis of their compatibility with the reactionscheme, can be prepared in the manner of Scheme VIII by acylation of theamide intermediate (30) of Scheme XI with an acylating agent of formula(21), as shown in Scheme XII.

The subject compounds of the present invention were tested forbiological activity according to the following procedures.

Vasopressin Binding in Chinese Hamster Ovary Cell Membranes ExpressingHuman Vasopressin V_(1a) Subtype Receptors

Receptor Source:

Chinese hamster ovary cells (CHO cells) stably transfected with thehuman vasopressin V_(1a) subtype receptors were either obtained fromBiosignal Inc., 1744 rue Williams, Montreal, Quebec, Canada or obtainedfrom M. Thibonnier, Case Western Reserve University School of Medicine,Cleveland, Ohio.

A. Passaging and Amplification of Cells:

CHO cells transfected with the human vasopressin V_(1a) subtypereceptors obtained from M. Thibonnier (pZeoSV vector) are allowed togrow to confluency (approx. >90%) in T-150 flasks under sterileconditions, in a cell culture medium of F-12 Nutrient Mixture (HAM) withL-glutamine (Gibco Cat. #11765-054) containing 15 mM HEPES (Gibco Cat.#15630-080), 1% antibiotic/antimycotic (add 5 mL 100×, Gibco Cat.#15240-062 per 500 mL F-12), 250 μg/mL Zeocin (add 1.25 mL of 100 mg/mLInvitrogen R-250-01 per 500 mL F-12) and 10% Fetal Bovine Serum(Qualified, heat inactivated, Gibco Cat. #16140-063). The medium isremoved by aspiration and the cells are washed with 10 mL of Hank'sBalanced Salt solution (Gibco Cat. #14175-095). The salt solution isremoved by aspiration and the cells are trypsinized with 5 mL oftrypsin-EDTA (0.05% trypsin, 0.53 mM EDTA-4Na, Gibco Cat. #25300-070)for 1 min. The trypsin is removed by aspiration and the cells dislodgedby tapping. Cell Culture medium (eg, 30 mL for 1:30 split) isimmediately added and mixed well to inactivate trypsin. 1 mL of detachedcells is added to new culture flasks containing fresh cell culturemedium (eg, into 25 mL per T-150 flask), and mixed gently. The cells areincubated at 37° C. in 5% CO₂. The medium is changed at 3 to 4 daysinterval (or as appropriate). The cells grow to confluency(approx. >75%-95%) within 7-8 days. All steps are done under sterileconditions.

B. Membrane Preparation:

The cells are washed twice gently with Hank's Balanced Salt solution(e.g,. use 10 mL per T-150 flask). The excess is removed and the cellsare bathed for 15-30 min. in an enzyme-free Cell Dissociation Buffer(e.g. use 8 mL Hank's Based, Gibco Cat. #13150-016 per T-150 flask)until the cells are loosened. The contents are transferred to centrifugetubes (50 mL size) kept in ice bath. All subsequent steps are done at 4°C. The tubes are centrifuged at 300×g for 15 min (1380 rpm on SORVAL,Model RT6000D, using rotor for 50 mL tubes). The supernatant isdiscarded and the cells suspended in homogeneizing buffer(10 mM Tris-HClcontaining 0.25 M sucrose and 1 mM EDTA, pH 7.4) ensuring that thevolume of the buffer is about ten times the volume of the cell pellet.The cells are pooled into a centrifuge tube (50 mL) and homogenized withPolytron at setting 6 for 10 sec. The homogenate is transferred into aPotter-Elvjehm homogenizer and homogenized with 3 strokes. Thehomogenate is centrifuged at 1500×g for 10 min at 4° C. (3100 rpm usingSORVAL, model RT6000D, using rotor for 50 mL tubes). The pellet isdiscarded. The supernatant is centrifuged at 100,000×g for 60 min. at 4°C. (Beckman L8-80M ultracentrifuge; spin at 37,500 rpm with rotor type70 Ti for 50 mL tubes; 38,000 rpm with type 80Ti for 15 mL tubes; or35,800 rpm with rotor type 45Ti). The supemantant is discarded and thepellet suspended in 3 to 4 mL of Tris buffer (50 mM TRIS-HCl, pH 7.4).The protein content is estimated by the Bradford or Lowry method. Thevolume of the membrane suspension is adjusted with the membrane buffer(50 mM Tris-HCl containing 0.1% BSA and 0.1 mM PMSF) to give 3.0 mg/mL(or as appropriate) of protein. The membranes are aliquoted and storedat −70° C.

C. Radioligand Binding Assay:

In wells of a 96-well format microtiter plate, is added 90, 110 or 130μL (to make up a final volume of 200 μL) of assay buffer containing 50mM of Tris-HCl (pH 7.4), BSA (heat inactivated, protease-free), 0.1% of5 mM MgCl₂, 1 mg % aprotinin, 1 mg % leupeptin, 2 mg %1,10-phenanthroline, 10 mg % trypsin inhibitor, and 0.1 mM PMSF. Theinhibitors are added on the day of the experiment. The components aremixed at room temperature, and then kept in ice bath followingadjustment of the pH to 7.4. To each well is added 20 μL of unlabeledManning ligand (to give a final concentration of 0.1 to 10 nM forstandard curve and 1000 nM for non specific binding) or test compoundsin 50% DMSO (e.g. for final concentrations of 0.1 to 1000 nM or asappropriate) or 50% DMSO as vehicle control. 20 μL of 50% DMSO is addedfor Manning and other peptide ligands and the assay buffer volume isadjusted accordingly. To each well is added 50 μL of frozen membranesuspension thawed immediately prior to use and diluted in the assaybuffer to the required concentration (equivalent to 25 to 50 μg ofprotein/well as needed). 20 μL of 8 nM [³H]Manning ligand in the assaybuffer, prepared just before use, is added, and incubated at roomtemperature for 60 min. shaking the plate on a mechanical shaker for thefirst 15 min. The incubation is stopped by rapid filtration of the theplate contents followed by wash with ice-cold buffer (50 mM Tris-HCl, pH7.4) using a cell harvester (Tomtek and Printed filtermat-B filterpaper). The filter paper is thoroughly dried (7-12 min. in a microwaveoven) and impregnated with MeltiLex B/H melt-on scintillation wax sheetsand the radioactivity counted in a betaplate scintillation counter.

Vasopressin Binding in Chinese Hamster Ovary Cell Membranes ExpressingHuman Vasopressin V₂ Subtype Receptors

Receptor Source:

Chinese Hamster Ovary (CHO) cells stably transfected with the human V₂subtype receptors were obtained from M. Thibonnier, Case Western ReserveUniversity School of Medicine, Cleveland, Ohio.

A. Passaging and Amplification of Cells:

CHO cells transfected with the human vasopressin V₂ subtype receptorsobtained from M. Thibonnier (pZeoSV vector) are allowed to grow toconfluency (approx. >90%) in T-150 flasks under sterile conditions, in acell culture medium of F-12 Nutrient Mixture (HAM) with L-glutamine(Gibco Cat. #11765-054) containing 15 mM HEPES (Gibco Cat. #15630-080),1% antibiotic/antimycotic (add 5 mL 100×, Gibco Cat. #15240-062 per 500mL F-12), 250 μg/mL Zeocin (add 1.25 mL of 100 mg/mL Invitrogen R-250-01per 500 mL F-12) and 10% Fetal Bovine Serum (Qualified, heatinactivated, Gibco Cat. #16140-063). The medium is removed by aspirationand the cells washed with 10 mL of Hank's Balanced Salt solution (GibcoCat. #14175-095). The salt solution is removed by aspiration and thecells trypsinized with 5 mL of trypsin-EDTA (0.05% trypsin, 0.53 mMEDTA-4Na, Gibco Cat. #25300-070) for 1 min. The trypsin is removed byaspiration and the cells dislodged by tapping. Cell Culture medium (e.g.30 mL for 1:30 split) is immediately added and mixed well to inactivatetrypsin. 1 mL of detached cells is added to new culture flaskscontaining fresh Cell Culture medium (e.g. into 25 mL per T-150 flask),and mixed gently. The cells are incubated at 37° C. in 5% CO₂. Themedium is changed at 3 to 4 day interval (or as appropriate). The cellsgrow to confluency (approx. >75%-95%) within 7-8 days. All steps aredone under sterile conditions.

B. Membrane Preparation:

The cells are washed twice gently with Hank's Balanced Salt solution(e.g. use 10 mL per T-150 flask). The excess solution is removed and thecells bathed for 15-30 min. in an enzyme-free Cell Dissociation Buffer(e.g. use 8 mL Hank's Based, Gibco Cat. #13150-016 per T-150 flask)until cells are loosened. The contents are transferred to centrifugetubes (50 mL size) kept in ice bath. All subsequent steps are done at 4°C. The tubes are centrifuged at 300×g for 15 min (1380 rpm on SORVAL,Model RT6000D, using rotor for 50 mL tubes). The supernatant isdiscarded and the cells suspended in homogeneizing buffer(10 mM Tris-HClcontaining 0.25 M sucrose and 1 mM EDTA, pH 7.4) ensuring that thevolume of the buffer is about ten times the volume of the cell pellet.The cells are pooled into a centrifuge tube (50 mL) and homogenized withPolytron at setting 6 for 10 sec. The homogenate is transferred into aPotter-Elvjehm homogeneizer and homogenized with 3 strokes. Thehomogenate is centrifuged at 1500×g for 60 min at 4° C. (3100 rpm usingSORVAL, model RT6000D, using rotor for 50 mL tubes). The pellet isdiscarded. The supernatant is centrifuged at 100,000×g for 60 min. at 4°C. (Beckman L8-80M ultracentrifuge; spin at 37,500 rpm with rotor type70 Ti for 50 mL tubes; 38,000 rpm with type 80Ti for 15 mL tubes; or35,800 rpm with rotor type 45Ti). The supernantant is discarded and thepellet suspended in 3 to 4 mL of Tris buffer (50 mM TRIS-HCl, pH 7.4).The protein content is estimated by the Bradford or Lowry method. Thevolume of the membrane suspension is adjusted with the membrane buffer(50 mM Tris-HCl containing 0.1% BSA and 0.1 mM PMSF) to give 3.0 mg/mL(or as appropriate) of protein. The membranes are aliquoted and storedat −70° C.

C. Radioligand Binding Assay:

In wells of a 96-well format microtiter plate, is added 90, 110 or 130μL (to make up a final volume of 200 μL) of assay buffer containing 50mM of Tris-HCl (pH 7.4), BSA (heat inactivated, protease-free), 5 mM of0.1% MgCl₂, 1 mg % aprotinin, 1 mg % leupeptin, 2 mg %1,10-phenanthroline, 10 mg % trypsin inhibitor, and 0.1 mM PMSF. Theinhibitors are added on the day of the experiment. The components aremixed at room temperature, and then kept in ice bath followingadjustment of the pH to 7.4. To each well is added 20 μL of unlabeledarginine vasopressin (AVP) (to give a final concentration of 0.1 to 10nM for standard curve and 1000 nM for non specific binding) or testcompounds in 50% DMSO (e.g. for final concentrations of 0.1 to 1000 nMor as appropriate) or 50% DMSO as vehicle control. For vasopressin andother peptide ligands is added 20 μL of 50% DMSO and the assay buffervolume is adjusted accordingly. To each well is added 50 μL of frozenmembrane suspension thawed immediately prior to use and diluted in assaybuffer to the required concentration (equivalent to 25 to 50 μg ofprotein/well as needed). 20 μL of 8 nM[³H]arginine vasopressin (AVP)ligand in the assay buffer, prepared just before use is added andincubated at room temperature for 60 min. shaking the plate on amechanical shaker for the first 15 min. The incubation is stopped byrapid filtration of the plate contents followed by wash with ice-coldbuffer (50 mM Tris-HCl, pH 7.4) using a cell harvester (Tomtek andPrinted filtermat-B filter paper). The filter paper is thoroughly dried(7-12 min. in a microwave oven) and impregnated with MeltiLex B/Hmelt-on scintillation wax sheets and the radioactivity counted in abetaplate scintillation counter.

Oxytocin binding in Chinese Hamster Ovary cell membranes expressinghuman oxytocin receptors

Receptor Source:

Chinese Hamster Ovary (CHO) cells stably transfected with the humanoxytocin (cf. T. Kimura et al., U.S. Pat. No. 5,466,584 (1995) to RohtoPharmaceutical Co. Ltd., Osaka, Japan) were obtained from Dr. M.Thibonnier, Case Western Reserve University School of Medicine,Cleveland, Ohio.

A. Passaging and Amplification of Cells:

CHO cells transfected with the human oxytocin receptors obtained from M.Thibonnier (pcDNA3.1 vector) are allowed to grow to confluency(approx. >90%) in T-150 flasks under sterile conditions, in a cellculture medium of F-12 Nutrient Mixture (HAM) with L-glutamine (GibcoCat. #11765-054) containing 15 mM HEPES (Gibco Cat. #15630-080), 1%antibiotic/antimycotic (add 5 mL 100×, Gibco Cat. #15240-062 per 500 mLF-12), 400 μg/mL of Geneticin (add 4 mL of 50 mg/mL per 500 mL F-12) and10% Fetal Bovine Serum (Qualified, heat inactivated, Gibco Cat.#16140-063). The medium is removed by aspiration and the cells arewashed with 10 mL of Hank's Balanced Salt solution (Gibco Cat.#14175-095). The salt solution is removed by aspiration and the cellstrypsinized with 5 mL of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA-4Na,Gibco Cat. #25300-070) for 1 min The trypsin is removed by aspirationand the cells dislodged by tapping. Cell Culture medium (e.g. 30 mL for1:30 split) is immediately added and mixed well to inactivate trypsin. 1mL of detached cells is added to new culture flasks containing freshCell Culture medium (e.g. into 25 mL per T-150 flask), and mixed gently.The cells are incubated at 37° C. in 5% CO₂. The medium is changed at 3to 4 days interval (or as appropriate). The cells grow to confluency(approx. >75%-95%) within 7-8 days. All steps are done under sterileconditions.

B. Membrane Preparation:

The cells are washed twice gently with Hank's Balanced Salt solution(eg, use 10 mL per T-150 flask). The excess is removed and the cellsbathed for 15-30 min. in an enzyme-free Cell Dissociation Buffer (eg,use 8 mL Hank's Based, Gibco Cat. #13150-016 per T-150 flask) untilcells are loosened. The contents are transferred to centrifuge tubes (50mL size) kept in ice bath. All subsequent steps are done at 4° C. Thetubes are centrifuged at 300×g for 15 min (1380 rpm on SORVAL, ModelRT6000D, using rotor for 50 mL tubes). The supernatant is discarded andthe cells suspended in homogenizing buffer (10 mM Tris-HCl containing0.25 M sucrose and 1 mM EDTA, pH 7.4) ensuring that the volume of thebuffer is about ten times the volume of the cell pellet. The cells arepooled into a centrifuge tube (50 mL) and homogenized with Polytron atsetting 6 for 10 sec. The homogenate is transferred into aPotter-Elvjehm homogenizer and homogenized with 3 strokes. Thehomogenate is centrifuged at 1500×g for 10 min at 4° C. (3100 rpm usingSORVAL, model RT6000D, using rotor for 50 mL tubes). The pellet isdiscarded. The supernatant is centrifuged at 100,000×g for 60 min. at 4°C. (Beckman L8-80M ultracentrifuge; spin at 37,500 rpm with rotor type70 Ti for 50 mL tubes; 38,000 rpm with type 80Ti for 15 mL tubes; or35,800 rpm with rotor type 45Ti). The supernantant is discarded and thepellet suspended in 3 to 4 mL of Tris buffer (50 mM TRIS-HCl, pH 7.4).The protein content is estimated by the Bradford or Lowry method. Thevolume of the membrane suspension is adjusted with the membrane buffer(50 mM Tris-HCl containing 0.1% BSA and 0.1 mM PMSF) to give 3.0 mg/mL(or as appropriate) of protein. The membranes are aliquoted and storedat −70° C.

C. Radioligand Binding Assay:

In wells of a 96-well format microtiter plate, is added 90, 110 or 130μL (to make up a final volume of 200 μL) of assay buffer containing 50mM of Tris-HCl (pH 7.4), BSA (heat inactivated, protease-free), 5 mM of0.1% MgCl₂, 1 mg % aprotinin, 1 mg % leupeptin, 2 mg %1,10-phenanthroline, 10 mg % trypsin inhibitor, and 0.1 mM PMSF. Theinhibitors are added on the day of the experiment. The components aremixed at room temperature, and then kept in ice bath followingadjustment of the pH to 7.4. To each well is added 20 μL of unlabeledoxytocin (to give a final concentration of 0.1 to 10 nM for standardcurve and 1000 nM for non specific binding) or test compounds in 50%DMSO (e.g. for final concentrations of 0.1 to 1000 nM or as appropriate)or 50% DMSO as vehicle control. For oxytocin and other peptide ligands20 μL of 50% DMSO is added and the assay buffer volume is adjustedaccordingly. To each well is added 50 μL of frozen membrane suspensionthawed immediately prior to use and diluted in assay buffer to therequired concentration (equivalent to 25 to 50 μg of protein/well asneeded). 20 μL of 8 nM [³H]oxytocin in the assay buffer, prepared justbefore use is added and incubated at room temperature for 60 min.shaking the plate on a mechanical shaker for the first 15 min. Theincubation is stopped by rapid filtration of the the plate contentsfollowed by wash with ice-cold buffer (50 mM Tris-HCl, pH 7.4) using acell harvester (Tomtek and Printed filtermat-B filter paper). The filterpaper is thoroughly dried (7-12 min. in a microwave oven) andimpregnated with MeltiLex B/H melt-on scintillation wax sheets and theradioactivity counted in a betaplate scintillation counter.

Binding data is either reported as percent inhibition at a certainconcentration or if an IC₅₀ was calculated, as a nanomolarconcentration.

The Results of these Tests on Representative Compounds of this Inventionare Shown in Table I.

TABLE 1 Binding to membranes of Chinese Hamster Ovary (CHO) cell linestably transfected with human vasopressin V_(1a) receptor subtype, humanV₂ vasopressin receptor subtype and human oxytocin receptor OT V_(1a) %inhibition @ V₂ Ex- % inhibition @ 100 nM % inhibition @ ample 100 nM(IC₅₀, nM)* (IC₅₀, nM)* 100 nM (IC₅₀, nM)*  1  (0.98)  879 (1557)  1G 21   0   4  2 (19.6) (2188) (4588)  3  (4.92) (1458) (2622)  4  (2.07) (613) (1494) 56  (2.15)  (181.87) (2108.9)  5D  10   0   1  5E  26   8  15  6  (0.96)  (62.4)  (558)  7  99   62   14  8 (13)  (315) (>3000) 9  23   6   11 10  54   14   15 11 (76.17)   6   10 12 (47.07)   16   913  63   5   9 14  50   6   11 15  (3.19)  (645.54)  (120.52) 15B  19  −2   40 16  98   6   46 17  88   31   7 *Binding in Chinese HamsterOvary cell membranes expressing human vasopressin V_(1a) and V₂ subtypereceptors and human oxytocin receptorsThe following examples are presented to illustrate rather than limit thescope of this invention.

EXAMPLE 11-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol

Step A. 4-Amino-5-chloro-2-methoxy-benzoic acid methyl ester

4-Amino-5-chloro-2-methoxy benzoic acid (50.0 g, 248 mmol) was suspendedin methanol (500 mL) and the slurry cooled to 0° C. Thionyl chloride(54.3 mL, 744 mmol) was then added dropwise over the course of 20minutes. The initially clear solution turned to a white suspension. Thereaction was warmed to room temperature and stirred for 3 hours. Themethanol was evaporated and the resulting slurry suspended in diethylether (1 L). The solid was filtered and rinsed thoroughly with diethylether to afford the title compound (50.9 g) as the hydrochloride salt.The salt was suspended in 1 N sodium hydroxide and stirred vigorouslyfor 30 minutes. Filtration and thorough rinsing with water afforded thetitle compound free base as a white solid, m.p. 136-137° C.

¹H NMR (DMSO-d₆,400 MHz): δ 7.57 (s, 1H), 6.43 (s, 1H), 6.14 (s, 2H),3.71 (s, 3H), 3.67 (s, 3H).

Anal. Calcd. for C₉ H₁₀ClNO₃: C, 50.13, H, 4.67, N, 6.50. Found: C,49.85, H, 4.46, N, 6.65

MS [(+)-ACPl, m/z]: 216 [M+H]⁺. Calc'd for C₉H₁₁ClNO₃: 216.0428.

Step B. 5-Chloro-4-iodo-2-methoxy-benzoic acid methyl ester

4-Amino-5-chloro-2-methoxy benzoic acid methyl ester of Step A (5.00 g,23.2 mmol) was suspended in water (52 mL) and concentrated sulfuric acid(13 mL) was added. The resulting suspension was cooled to −1° C. and asolution of sodium nitrite (1.76 g, 25.5 mmol) in water (10 mL) wasadded at a rate which maintained the temperature below 0° C., resultingin the formation of a clear yellow solution. A mixture of potassiumiodide (4.23 g, 25.5 mmol) and iodine (3.24 g, 12.8 mmol) in water (50mL) was then added dropwise and the reaction stirred at 0° C. for 1.5hours. The reaction mixture was warmed to room temperature and extractedwith ethyl acetate. The combined extracts were washed sequentially with1 M aqueous sodium thiosulfate, 1 N sodium hydroxide and brine, anddried over anhydrous magnesium sulfate, filtered and concentrated,whereupon the product crystallized. The resulting orange crystals weresuspended in petroleum ether, filtered and dried in vacuo to provide thetitle compound (6.38 g), m.p. 72-73° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.72 (s, 1H), 7.66 (s, 1H), 3.83 (s, 3H),3.77 (s, 3H).

Anal. Calc'd for C₉H₈ClIO₃: C, 33.11, H, 2.47. Found: C, 33.21, H, 2.23.

MS [(+)-ACPl, m/z): 327 [M+H]⁺. Calcd. for C₉H₉ClIO₃: 326.9285.

Step C. 5-Chloro-4-iodo-2-methoxy-benzoic acid

A mixture od 5-chloro-4-iodo-2-methoxy benzoic acid methyl ester of StepB (3.00 g, 9.19 mmol) and sodium hydroxide (1.10 g, 27.6 mmol) inmethanol (92 mL) was refluxed for 12 hours. The reaction was cooled toroom temperature and the solvent evaporated. The residue was dissolvedin 1 N sodium hydroxide (75 mL), the solution washed with diethyl etherand the organic washings discarded. The aqueous phase was acidified with2 N hydrochloric acid and extracted with diethyl ether. The combinedextracts were dried over anhydrous sodium sulfate, filtered andconcentrated to afford the title carboxylic acid (2.64 g) as orangecrystals, m.p. 150-151° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 13.03 (br, 1H), 7.70 (s, 1H), 7.63 (s, 1H),3.82 (s, 3H).

Anal. Calcd. for C₈H₆ClIO₃: C, 30.75, H, 1.94. Found: C, 31.28, H, 1.78.

MS [(−)-ACPl, m/z]: 311 [M−H]⁻. Calcd. for C₈H₅ClIO₃: 310.8972.

Step D.(5H,11H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl)-(5-chloro-4-iodo-2-methoxy-phenyl)-methanone

To a mixture of 5-chloro-4-iodo-2-methoxy benzoic acid of Step C (0.900g, 2.88 mmol) and N,N-dimethylformamide (6.7 mL, 86.4 mmol) in anhydrousdichloromethane (14.4 mL) was added dropwise oxalyl chloride (0.263 mL,3.02 mmol). The mixture was heated to reflux for 1 hour, then cooled toroom temperature and evaporated to dryness. Fresh anhydrousdichloromethane (25 mL) was added, the resulting solution wasconcentrated and the residue dried in vacuo. The crude acid chloridethus obtained and 10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine(0.584 g, 3.17 mmol) were combined in anhydrous dichloromethane(14.4mL), and N,N-diisopropylethylamine (0.447 mL, 3.46 mmol) was added.After stirring at room temperature for 18 hours, the reaction mixturewas diluted with dichloromethane (15 mL) and washed sequentially with 1N hydrochloric acid, 1 N sodium hydroxide, and brine. The organic phasewas dried over anhydrous magnesium sulfate, filtered and concentrated toafford the crude title amide which was recrystallized from diethyl etherto provide 1.23 g of pale orange crystals, m.p. 191-192° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.60-7.28 (m, 3H), 7.14-7.01 (m, 3H), 6.79(s, 1H), 5.95 (s, 1H), 5.89 (t, 1H), 5.15 (br, 4H), 3.56 (s, 3H).

Anal. Calcd. for C₂₀H₁₆ClIN₂O₂: C, 50.18, H, 3.37, N, 5.85. Found: C,50.47, H, 3.28, N, 5.74.

MS (El, m/z): 478 [M]⁺. Calcd. for C₂₀H₁₆ClIN₂O₂: 477.9946.

Step E.(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[5-chloro-2-methoxy-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-methanone

(5H,11H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl)-(5-chloro-4-iodo-2-methoxy-phenyl)-methanone of Step D(0.500 g, 1.04 mmol), bis(pinacolato)diboron (0.289 g, 1.14 mmol),potassium acetate (0.306 g, 3.12 mmol) anddichloro[1,1′-bis(diphenylphosphino) ferrocene]palladium (II)dichloromethane adduct (0.025 g, 0.031 mmol) were combined in anhydrousdimethyl sulfoxide (5.2 mL) and heated to 80° C. overnight. The reactionmixture was cooled to room temperature, diluted with ethyl acetate andwashed with water and brine. The organic phase was dried over anhydroussodium sulfate, diluted with hexane and filtered through a plug ofsilica gel. The filtrate was concentrated to an oil which crystallizedfrom diethyl ether/petroleum ether (−20° C.). to provide the titlecompound (0.430 g) as white crystalline solid, m.p. 92-98° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.48-7.36 (m, 2H), 7.12-7.03 (m, 4H), 6.79(s, 1H), 5.95 (m, 1H), 5.89 (t, 1H), 5.20 (br, 4H), 3.48 (br, 3H), 1.26(s, 12H).

Anal. Calcd. for C₂₆H₂₈BClN₂O₄: C, 56.22, H, 5.89, N, 5.85. Found: C,56.23, H, 5.63, N, 6.24.

MS [(+)-ESI, m/z]: 479 [M+H]⁺. Calcd. for C₂₆H₂₉BClN₂O₄: 479.1910.

Step F.10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[5-chloro-2-methoxy-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-methanoneof Step E (0.220 g, 0.459 mmol), cyclohex-1-en-1-yltrifluoromethanesulfonate (0.116 g, 0.505 mmol) anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (0.011 g, 0.014 mmol) were combined inN,N-dimethylformamide (2.3 mL). Aqueous sodium carbonate (2 M, 1.15 mL,2.30 mmol) was added and the reaction was heated to 60° C. for 2 hours.After cooling to room temperature, the reaction mixture was diluted withethyl acetate and washed with water and brine. The organic phase wasdried over anhydrous magnesium sulfate, filtered and concentrated. Flashcolumn chromatography on silica gel eluting with a solvent gradient from30 to 40% of ethyl acetate in hexane, afforded the title compound (0.140g) as an oil. The oil was dissolved in diethyl ether/petroleum ether andconcentrated to afford a white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.38 (d, 2H), 7.11 (t, 1H), 7.06-7.00 (m,2H), 6.79 (s, 1H), 6.57 (s, 1H), 5.95 (s, 1H), 5.89 (t, 1H), 5.55 (s,1H), 5.24-4.60 (m, 4H), 3.52 (s, 3H), 2.13-2.09 (m, 4H), 1.68-1.57 (m,4H).

Anal. Calcd. for C₂₆H₂₅ClN₂O₂+0.03 C₄H₁₀O: C, 71.76, H, 5.79, N, 6.44.Found: C, 71.66H, 5.59, N, 6.10.

MS [(+)-APC, m/z): 433 [M+H]⁺. Calcd. for C₂₆H₂₆ClN₂O₂: 433.1684

Step G.10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid

10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepineof Step F (0.300 g, 0.693 mmol), and N,N-diisopropylethylamine (0.127mL, 0.728 mmol) were dissolved in anhydrous dichloromethane (2.8 mL).Trichloroacetyl chloride (0.116 mL, 1.04 mmol) was added dropwise andthe reaction stirred at room temperature for 3 hours. The mixture wasdiluted with ethyl acetate and washed with 1 N hydrochloric acid, 1 Nsodium hydroxide and brine. The organic phase was dried over anhydrousmagnesium sulfate, diluted with hexane (50 mL) and filtered throughsilica gel eluting with 30% ethyl acetate in hexane. Concentration ofthe filtrate afforded crude trichloroacetate (0.360 g). This materialwas dissolved in acetone (4.2 mL) and 2.5 N sodium hydroxide (0.750 mL)was added. The reaction was stirred at room temperature for 4 hours,then diluted with 1 N hydrochloric acid (50 mL) and extracted withdiethyl ether. The organic phase was extracted with 1 N sodium hydroxideand the combined basic extracts acidified with 2 N hydrochloric acid.The aqueous phase was extracted with diethyl ether and the extract driedover anhydrous sodium sulfate, filtered and concentrated to afford thetitle compound (0.280 g) as a white solid, m.p. 192° C. (dec.)

¹H NMR (DMSO-d₆, 400 MHz): δ 12.34 (br, 1H), 7.42 (br, 1H), 7.25 (d,1H), 7.07 (t, 1H), 6.98 (t, 1H), 6.93 (d, 1H), 6.72 (d, 1H), 6.54 (br,1H), 6.10 (d, 1H), 5.90-4.60 (m, 5H), 3.47 (br, 3H), 2.14-2.09 (m, 4H),1.65-1.57 (m, 4H).

Anal. Calcd. for C₂₇H₂₅ClN₂O₄: C, 67.99, H, 5.28, N, 5.87. Found: C,67.71, H, 5.23, N, 5.49.

MS [(−)-ACPl, m/z]: 475 [M−H]⁻. Calcd. for C₂₇H₂₄ClN₂O₄: 475.1426.

Step H.1-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol

10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Step G (0.120 g, 0.252 mmol), N-methyl-D-glucamine (0.059 g,0.302 mmol), 1-hydroxy benzotriazole (0.037 g, 0.277 mmol) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.053 g,0.277 mmol) were combined in amine-free N,N-dimethylformamide (1.0 mL),followed by addition of N,N-diisopropylethylamine (0.066 mL, 0.378mmol). The reaction was stirred at room temperature for 12 hours, thendiluted with ethyl acetate and washed with 1 N hydrochloric acid, 1 Nsodium hydroxide and brine. The combined aqueous washings were saturatedwith sodium chloride and extracted with ethyl acetate. The organicphases were combined, dried over anhydrous magnesium sulfate, filteredand concentrated. The residue was purified by flash columnchromatography on silica gel eluting with 10% methanol in chloroform, toafford 0.110 g of the title compound as a white solid.

¹H NMR (DMSO-d₆+D₂O; 400 MHz): δ 7.46-7.29 (m, 2H), 7.07 (t, 1H), 6.96(t, 1H), 6.90 (d, 1H), 6.50 (s, 1H), 6.31 (s, 1H), 6.01 (s, 1H), 5.52(s, 1H), 5.22 (s, 2H), 3.90 (br, 2H), 3.61-3.34 (m, 9H), 3.05 (br, 3H),2.11-2.05 (m, 4H), 1.61-1.50 (m, 4H).

Anal. Calcd. for C₃₄H₄₀ClN₃O₈: C, 62.43, H, 6.16, N, 6.42. Found: C,62.25, H, 6.41, N, 6.15.

MS [(+)-ESI, m/z]: 654 [M+H]⁺. Calcd. for C₃₄H₄₁ClN₃O₈: 654.2584.

EXAMPLE 210-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

1-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitolof Example 1 (0.140 g, 0.214 mmol), was dissolved in anhydroustetrahydrofuran (2.1 mL) and sodium hydride (0.051 g, 2.14 mmol) added.The mixture was stirred at room temperature until gas evolution ceased(approximately 15 minutes), followed by addition of methyl iodide (0.266mL, 4.28 mmol). After stirring at room temperature for 72 hours, thereaction was quenched with saturated aqueous ammonium chloride (50 mL)and extracted with ethyl acetate. The combined extracts were dried overanhydrous magnesium sulfate, filtered and concentrated. The residue waspurified by flash column chromatography on silica gel eluting with 5%methanol in chloroform, to provide 0.140 g of the title compound as awhite solid.

¹H NMR (DMSO-d₆+D₂O; 400 MHz):

□.41-7.30 (m, 2H), 7.08 (t, 1H), 6.99 (t, 1H), 6.94 (d, 1H), 6.54 (s,1H), 6.29 (d, 1H), 6.04 (s, 1H), 5.55 (s, 1H), 5.29 (br, 2H), 3.68-3.10(m, 29H), 2.13-2.09 (m, 4H), 1.64-1.58 (m, 4H).

Anal. Calcd for C₃₉H₅₀ClN₃O₈: C, 64.67, H, 6.96, N, 5.80. Found: C,64.68, H, 7.01, N, 5.71.

MS [(+)—ACPl, m/z]: 724 [M+H]⁺. Calcd for C₃₉H₅₁ClN₃O₈: 724.3367.

EXAMPLE 310-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

1-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitolof Example 1 (0.190 g, 0.290 mmol), and N,N-diisopropylethylamine (0.202mL, 1.16 mmol) were dissolved in anhydrous dichloromethane (5.8 mL) andthe solution cooled to 0° C. N,N′-Carbonyldiimidazole (0.094 g, 0.580mmol) was added and the reaction stirred at 0° C. for 1 hour, thenwarmed to room temperature and stirring continued for 1.5 hours. Thesolution was diluted with dichloromethane and washed with 0.1 Nhydrochloric acid and saturated aqueous sodium bicarbonate. The organicphase was dried over anhydrous sodium sulfate, filtered andconcentrated. Purification of the residue by flash column chromatographyon silica gel eluting with a solvent gradient from 0 to 10% of methanolin chloroform, afforded 0.190 g of the title compound as a white,partially crystalline solid, m.p. 163-176° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.50-7.31 (m, 2H), 7.08 (t, 1H), 7.02 (t,1H), 6.94 (d, 1H), 6.57 (br, 1H), 6.40 (d, 1H), 6.36 (br, 1H), 6.07 (d,1H), 5.55 (br, 1H), 5.39-5.20 (m, 4H), 4.96-4.90 (m, 1H), 4.87 (q, 1H),4.74 (br, 1H), 4.57 (t, 1H), 4.41 (dd, 1H), 4.05-3.98 (m, 2H), 3.82-3.79(m, 1H), 3.49 (br, 3H), 3.15 (s, 3H), 2.14-2.08 (m, 4H), 1.69-1.57 (m,4H).

Anal. Calcd. for C₃₆H₃₆ClN₃O₁₀: C, 61.23, H, 5.14, N, 5.95. Found: C,60.84, H, 5.05, N, 5.79.

MS [(+)-ESI, m/z]: 706 [M+H]⁺, 728 [M+Na]⁺. Calcd. for C₃₆H₃₇ClN₃O₁₀:706.2169.

EXAMPLE 410-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-(2,3-dihydroxypropyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylic acid of Example 1, Step G (0.370g, 0.776 mmol), 3-methylamino-1,2-propanediol (0.098 g, 0.931 mmol),1-hydroxy benzotriazole (0.115 g, 0.854 mmol) and1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.164 g,0.854 mmol) were combined in amine-free N,N-dimethylformamide (3.9 mL),followed by addition of N,N-diisopropylethylamine (0.202 mL, 1.16 mmol).The reaction was stirred at room temperature for 12 hours, then dilutedwith ethyl acetate and washed with 1 N hydrochloric acid, 1 N sodiumhydeoxide and brine. The combined aqueous washings were saturated withsodium chloride and extracted with ethyl acetate. The combined organicphases were dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography onsilica gel eluting with a solvent gradient from 0 to 5% of methanol inchloroform, to afford 0.400 g of the title compound as a white,crystalline solid, m.p.155-157° C.

¹H NMR (DMSO-d₆+D₂O; 400 MHz): δ 7.44-7.27 (m, 2H), 7.07 (t, 1H), 6.96(t, 1H), 6.89 (d, 1H), 6.49 (s, 1H), 6.28 (d, 1H), 6.01 (s, 1H), 5.51(s, 1H), 5.20 (br, 2H), 4.74-4.58 (m, 2H), 3.82 (br, 1H), 3.62-3.32 (m,7H), 3.04 (br, 3H), 2.09-2.05 (m, 4H), 1.60-1.53 (m, 4H).

Anal. Calcd. for C₃₁H₃₄ClN₃O₅: C, 66.01, H, 6.08, N, 7.45. Found: C,65.69, H, 5.99, N, 7.34.

MS [(+)-ESI, m/z]: 564 [M+H]⁺. Calcd. for C₃₁H₃₅ClN₃O₅: 564.2267.

EXAMPLE 510-[4-(Cyclohex-1-en-1-yl)-3-methyl-benzoyl]-10,11-dohydro-5H-pyrrolo[2,1-c][1,4benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol

Step A.(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-(4-bromo-3-methyl-phenyl)-methanone0.20 hydrate

To a stirred mixture of 4-bromo-3-methyl benzoic acid (21.5 g, 100 mmol)and N,N-dimethylformamide (0.251 mL, 3.00 mmol) in anhydrousdichloromethane (200 mL) was added dropwise oxalyl chloride (9.16 mL,105 mmol). The mixture was heated to reflux for 1.5 hours, then cooledto room temperature and the solvent evaporated. Fresh anhydrousdichloromethane (200 mL) was added and the resulting solutionconcentrated and the residue was dried in vacuo. The crude acid chloridethus obtained and 10,11-dihydro-5H-pyrrolo[1,2-c][1,4]benzodiazepine(17.5 g, 95.0 mmol) were combined in anhydrous dichloromethane (200 mL),and N,N-diisopropylethylamine (19.2 mL, 110 mmol) was added. Afterstirring at room temperature for 18 hours, the reaction mixture waswashed with 1 N hydrochloric acid, 1 N sodium hydroxide and brine. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andconcentrated to afford the crude amide which was recrystallized fromethyl acetate to provide pale orange crystals (34.8 g) of the titlecompound, m.p.175-176° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.45 (dd, 1H), 7.38 (d, 1H), 7.33 (d, 1H),7.18 (dt, 1H), 7.10 (t, 1H), 6.92 (s, 1H), 6.90 (s, 1H), 6.82 (t, 1H),5.94 (s, 1H), 5.91 (t, 1H), 5.27-4.80 (br, 4H), 2.22 (s, 3H).

Anal. Calcd. for C₂₀H₁₇BrN₂O+0.20H₂O: C, 62.42, H, 4.56, N, 7.28. Found:C, 62.43, H, 4.60, N, 7.24.

MS [(+)-ESI, m/z]: 381 [M+H]⁺. Calcd. for C₂₀H₁₈BrN₂O: 381.0598.

Step B.(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[3-methyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-methanonesolvate with 0.12 ethyl acetate

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-(4-bromo-3-methyl-phenyl)-methanoneof Step A (20.0 g, 52.5 mmol), bis(pinacolato)diboron (14.7 g, 57.8mmol), potassium acetate (15.5 g, 158 mmol) anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (1.29 g, 1.58 mmol) were combined in anhydrousdimethyl sulfoxide (263 mL) and heated to 80° C. for 18 hours. Thereaction was cooled to room temperature and additional catalyst (1.29 g,1.58 mmol) and bis(pinacolato)diboron (3.33 g, 13.1 mmol) were added.Heating was resumed at 80° C. for an additional 18 hours. The reactionmixture was cooled to room temperature, diluted with ethyl acetate (500mL) and filtered through silica gel. The filtrate was washed with waterand brine. The organic phase was dried over anhydrous sodium sulfate,diluted with hexane and filtered through a plug of silica gel. Thefiltrate was concentrated to an oil and pentane added, causing theproduct to crystallize. The off-white crystals were filtered and driedin vacuo to provide 18.4 g of the title compound, m.p.190-193° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.45 (dd, 1H), 7.39 (d, 1H), 7.18-7.06 (m,3H), 6.98 (d, 1H), 6.91 (br, 1H), 6.81 (t, 1H), 5.94 (br, 1H), 5.91 (t,1H), 5.33-4.60 (br, 4H), 2.32 (s, 3H), 1.25 (s, 12H).

Anal. Calcd for C₂₆H₂₉BN₂O₃+O0.12 C₄H₈O₂: C, 72.46, H, 6.88, N, 6.38.Found: C, 70.80, H, 6.83, N, 6.06.

MS [(+)-ESI, m/z]: 429 [M+H]⁺. Calcd. for C₂₆H₃₀BN₂O₃: 429.2348.

Step C.(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-(4-cyclohex-1-en-1-yl-3-methyl-phenyl)-methanone

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[3-methyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-methanoneof Step B (3.50 g, 8.17 mmol), cyclohex-1-en-1-yltrifluoromethanesulfonate (2.26 g, 9.80 mmol) anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (0.200 g, 0.245 mmol) were combined inN,N-dimethylformamide (40.9 mL). Aqueous sodium carbonate (2 M, 20.5 mL,40.9 mmol) was added and the reaction heated to 60° C. overnight. Aftercooling to room temperature, the reaction mixture was diluted with ethylacetate and the organic layer washed with water and brine. The organicphase was dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was dissolved in hot ethyl acetate/petroleumether (1:1) and filtered. The filtrate was concentrated and the residuerecrystallized from petroleum ether to afford 2.52 μg of the titlecompound as light brown crystals, m.p. 182-183° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.47 (dd, 1H), 7.21-7.10 (m, 3H), 6.93 (d,2H), 6.83 (d, 1H), 6.81 (t, 1H), 5.93-5.91 (m, 2H), 5.43 (m, 1H), 5.26(br, 2H), 5.20-4.80 (br, 2H), 2.11 (s, 3H), 2.09-2.05 (m, 4H), 1.67-1.56(m, 4H).

Anal. Calcd. for C₂₆H₂₆N₂O+0.15H₂O: C, 81.07, H, 6.88, N, 7.27. Found:C, 81.03, H, 6.86, N, 7.24.

MS [(+)-ESI, m/z]: 383 [M+H]⁺. Calcd. for C₂₆H₂₇N₂O: 383.2128.

Step D.2,2,2-Trichloro-1-[10-(4-cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]-methanone

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-(4-cyclohex-1-en-1-yl-3-methyl-phenyl)-methanoneof Step C (1.03 g, 2.69 mmol), and N,N-diisopropylethylamine (0.937 mL,5.38 mmol) were dissolved in anhydrous dichloromethane (13.5 mL) andtrichloroacetyl chloride (0.901 mL, 8.07 mmol) added dropwise. Thereaction was stirred at room temperature for 3 hours, and the solventwas evaporated. The residue was diluted with ethyl acetate and filteredthrough a plug of silica gel. The filtrate was washed with 0.1 Nhydrochloric acid, saturated aqueous sodium bicarbonate, and brine,dried over anhydrous magnesium sulfate, filtered and concentrated. Theresidue was crystallized from ethyl acetate/hexane to afford 1.41 g ofthe title compound as white crystals, m.p. 149-150° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.46-7.43 (m, 2H), 7.21-7.16 (m, 2H), 7.12(dt, 1H), 6.95-6.90 (m, 2H), 6.85 (d, 1H), 6.34 (d, 1H), 5.95 (br, 2H),5.44 (m, 1H), 5.27 (br, 2H), 2.12 (s, 3H), 2.10-2.05 (m, 4H), 1.68-1.55(m, 4H).

Anal. Calcd. for C₂₈H₂₅C₁₃N₂O₂: C, 63.71, H, 4.77, N, 5.31. Found: C,63.35, H 4.62, N, 5.24.

MS [(+)-ESI, m/z]: 527.2 [M+H]⁺. Calcd. for C₂₈H₂₆C₁₃N₂O₂: 527.1058.

Step E.10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid

2.2.2-Trichioro-1-[10-(4-cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pvrrolo[2,1-c][1,4]benzodiazepin-3-yl]-ethanoneof Step D (0.700 g, 1.33 mmol), was dissolved in acetone (8.9 mL)followed by addition of 2.5 N sodium hydroxide (1.60 mL, 3.99 mmol). Thereaction was stirred at room temperature for 3 hours, and acidified with2 N hydrochloric acid. The acidic mixture was extracted with diethylether and the organic phase extracted with 1 N sodium hydroxide. Thecombined basic extracts were acidified with 2 N hydrochloric acid, andextracted with diethyl ether. The extract was dried over anhydroussodium sulfate, filtered and concentrated. The residue wasrecrystallized from diethyl ether to afford 0.450 g of the titlecompound as white crystals, 193° C. (dec.)

¹H NMR (DMSO-d₆, 400 MHz): δ 12.31 (s, 1H), 7.35 (dd, 1H), 7.17-7.13 (m,2H), 7.07 (dt, 1H), 6.91 (dd, 1H), 6.85 (t, 2H), 6.75 (d, 1H), 6.08 (d,1H), 5.92 (br, 2H), 5.43 (m, 1H), 5.14 (br, 2H), 2.11 (s, 3H), 2.10-2.05(m, 4H), 1.67-1.55 (m, 4H).

Anal. Calcd. for C₂₇H₂₆N₂O₃: C, 76.03, H, 6.14, N, 6.57. Found: C,75.71, H, 6.16, N, 6.48.

MS [(−)-ESI, m/z]: 425.2 [M−H]⁻. Calcd. for C₂₇H₂₅N₂O₃ 425.1862.

Step F.10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitolhemihydrate

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Step E (0.530 g, 1.24 mmol), N-methyl-D-glucamine (0.291 g, 1.49mmol), 1-hydroxy benzotriazole (0.184 g, 1.36 mmol) and1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.261 g,1.36 mmol) were combined in amine-free N,N-dimethylformamide (5.0 mL),followed by addition of N,N-diisopropylethylamine (0.324 mL, 1.86 mmol).The reaction was stirred at room temperature for 16 hours, then dilutedwith ethyl acetate and washed with 1 N hydrochloric acid, 1 N sodiumhydroxide and brine. The combined aqueous washings were saturated withsodium chloride and extracted with ethyl acetate. The organic phaseswere combined, dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography onsilica gel eluting with 8:1.8:0.2 chloroform/methanol/water to afford0.700 g of the title compound a white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.41 (dd, 1H), 7.17-7.13 (m, 2H), 7.08 (t,1H), 6.91-6.82 (m, 3H), 6.34 (s, 1H), 5.99 (s, 1H), 5.43 (m, 1H), 5.35(m, 2H), 4.91 (d, 1H), 4.51 (s, 1H), 4.40-4.32 (m, 3H), 3.92 (br, 1H),3.57-3.34 (m, 7H), 3.06 (br, 3H), 2.11 (s, 3H), 2.10-2.05 (m, 4H),1.67-1.55 (m, 4H).

Anal. Calcd. for C₃₄H₄₁N₃O₇+0.50H₂O: C, 66.65, H, 6.91, N, 6.86. Found:C 66.40, H 6.80, N6.76.

MS [(+)-ACPl, m/z]: 603.9 [M+H]⁺. Calcd. for C₃₄H₄₂N₃O₇: 604.3028.

EXAMPLE 610-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-((2S)-2-{(4R,5R)-5-[(1R)-1,2-dihydroxyethyl]-2-oxo-1,3-dioxolan-4-yl}-2-hydroxyethyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid methyl-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-hexyl]-amide ofExample 5 (0.190 g, 0.315 mmol), and N,N-diisopropylethylamine (0.110mL, 0.315 mmol) were dissolved in anhydrous dichloromethane (6.3 mL) andthe solution cooled to 0° C. N,N′-Carbonyldiimidazole (0.051 g, 0.315mmol) was added and the reaction stirred at 0° C. for 1 hour, thenwarmed to room temperature and stirring continued for 12 hours. Thesolution was diluted with ethyl acetate and washed with water, 0.1 Nhydrochloric acid and brine. The organic phase was dried over anhydrousmagnesium sulfate, filtered and concentrated. Purification of theresidue by flash column chromatography on silica gel eluting with ethylacetate, afforded 0.140 g of the title compound as a white, partiallycrystalline solid, m.p. 166-175° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.42 (d, 1H), 7.17-7.13 (m, 2H), 7.08 (t,1H), 6.92-6.82 (m, 3H), 6.35 (d, 1H), 6.05-6.02 (m, 1H), 5.76 (d, 1H),5.55-5.50 (m, 1H), 5.43-5.37 (m, 3H), 4.93-4.90 (m, 1H), 4.73-4.69 (m,2H), 3.90 (br, 1H), 3.79 (br, 1H), 3.59 (br, 1H), 3.49-3.24 (m, 3H),3.10 (s, 3H), 2.11 (s, 3H), 2.08-2.05 (m, 4H), 1.65-1.57 (m, 4H).

Anal. Calcd. for C₃₅H₃₉N₃O₈: C, 66.76, H, 6.24, N, 6.67. Found: C,66.53, H, 6.24, N, 6.41.

MS [(+)-ESI, m/z]: 630.2 [M+H]⁺. Calcd. for C₃₅H₄₀N₃O₈: 630.2817.

EXAMPLE 710-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid methyl-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-hexyl]-amide ofExample 5 (0.160 g, 0.265 mmol), and N,N-diisopropylethylamine (0.185mL, 1.06 mmol) were dissolved in anhydrous dichloromethane (5.3 mL) andthe solution cooled to 0° C. N,N′-Carbonyidiimidazole (0.086 g, 0.530mmol) was added and the reaction stirred at 0° C. for 1 hour, thenwarmed to room temperature and stirring continued for 0.75 hour. Thesolution was diluted with ethyl acetate and washed with water, 0.1 Nhydrochloric acid and brine. The organic phase was dried over anhydrousmagnesium sulfate, filtered and concentrated. Purification of theresidue by flash column chromatography on silica gel eluting with ethylacetate afforded 0.170 g of the title compound as a white powder, m.p.162-168° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.40 (d, 1H), 7.17-7.14 (m, 2H), 7.08 (t,1H), 6.93-6.82 (m, 3H), 6.40 (d, 1H), 6.35 (d, 1H), 6.05 (d, 1H),5.43-5.34 (m, 3H), 4.96-4.93 (m, 1H), 4.87 (q, 1H), 4.75 (s, 1H), 4.57(t, 1H), 4.42 (t, 1H), 4.05-3.98 (m, 2H), 3.81 (dd, 1H), 3.16 (s, 3H),2.11 (s, 3H), 2.08-2.05 (m, 4H), 1.66-1.57 (m, 4H).

Anal. Calcd. for C₃₆H₃₇N₃O₉: C, 65.94, H, 5.69, N, 6.41. Found: C,66.17, H, 6.01, N, 6.36

MS [(+)-ESI, m/z]: 656.1 [M+H]⁺. Calcd. for C₃₆H₃₈N₃O₉: 656.2609.

EXAMPLE 810-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid methyl-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-hexyl]-amide ofExample 5 (0.112 g, 0.186 mmol), was dissolved in anhydroustetrahydrofuran (1.9 mL) and sodium hydride (0.074 g, 1.86 mmol) added.The reaction was stirred at room temperature until gas evolution ceased(approximately 30 minutes), and then iodomethane (0.232 mL, 3.72 mmol)added. Stirring was continued for 72 hours, and the reaction mixturequenched with saturated aqueous ammonium chloride (30 mL). The productwas extracted with ethyl acetate and the combined extracts dried overanhydrous magnesium sulfate, filtered and concentrated. The residue waspurified by flash column chromatography on silica gel eluting with asolvent gradient from 0 to 5% of methanol in chloroform, to afford 0.100g of the title compound as a white solid, m.p. 68-83° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.38 (d, 1H), 7.17-7.14 (m, 2H), 7.08 (t,1H), 6.92-6.82 (m, 3H), 6.31 (d, 1H), 6.01 (d, 1H), 5.43 (s, 1H), 5.37(s, 2H), 5.26-4.85 (br, 2H), 3.69 (br, 2H), 3.58-3.54 (m, 1H), 3.39-3.24(m, 20H), 3.11 (s, 3H), 2.11 (s, 3H), 2.08-2.05 (m, 4H), 1.67-1.56 (m,4H).

Anal. Calcd. for C₃₉H₅₁N₃O₇: C, 69.52, H, 7.63, N, 6.24. Found: C,69.02, H, 7.66, N, 6.02.

MS [(+)-ACPl, m/z]: 674.4 [M+H]⁺. Calcd. for C₃₉H₅₂N₃O₇ 674.4512.

EXAMPLE 9(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionicacid ethyl ester

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Example 5, Step E (0.240 g, 0.563 mmol), L-tyrosine ethyl esterhydrochloride (0.157 g, 0.676 mmol), 1-hydroxy benzotriazole (0.084 g,0.619 mmol) and 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.119 g, 0.619 mmol) were combined in amine-freeN,N-dimethylformamide (2.25 mL), followed by the addition ofN,N-diisopropylethylamine (0.294 mL, 1.69 mmol). The reaction wasstirred at room temperature for 36 hours, then diluted with ethylacetate and washed with water, 1 N hydrochloric acid, saturated aqueoussodium bicarbonate, and brine. The combined aqueous washings weresaturated with sodium chloride and extracted with ethyl acetate. Thecombined organic phases were dried over anhydrous magnesium sulfate,filtered and concentrated. The residue was purified by flash columnchromatography on silica gel eluting with 50% ethyl acetate in hexane,followed by precipitation from diethyl ether with petroleum ether, toafford 0.207 g of the title compound as a white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 9.20 (s, 1H), 8.31 (d, 1H), 7.17 (d, 1H),7.10-6.99 (m, 5H), 6.88-6.78 (m, 3H), 6.68-6.63 (m, 3H), 6.03 (d, 1H),5.85-5.60 (br, 2H), 5.42 (m, 1H), 5.25-4.90 (br, 2H), 4.55-4.49 (m, 1H),4.10 (q, 2H), 3.02-2.89 (m, 2H), 2.10 (s, 3H), 2.08-2.04 (m, 4H),1.67-1.56 (m, 4H), 1.16 (t, 3H).

Anal. Calcd. for C₃₈H₃₉N₃O₅+0.15 C₄H₁₀O: C, 72.58, H, 6.25, N, 6.68.Found: C, 72.31, H, 6.63, N, 6.39.

MS [(+)-ACPl, m/z]: 618.3 [M+H]⁺. Calcd. for C₃₈H₄₀N₃O₅: 618.2968.

EXAMPLE 10(2S)-2-{[10-(4-Cyclohex-1-en-1-yl)-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionicacid

(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionicacid ethyl ester of Example 9 (0.120 g, 0.199 mmol), was dissolved inacetone (1.3 mL), followed by addition of 2.5 N sodium hydroxide (0.239mL, 0.597 mmol). The reaction was stirred at room temperature overnight,then acidified with 1 N hydrochloric acid. The acidic reaction mixturewas extracted with diethyl ether and the combined organic phasesextracted with 1 N sodium hydroxide. The combined aqueous extracts wereacidified with 2 N hydrochloric acid and extracted with diethyl ether.The combined extracts were dried over anhydrous sodium sulfate, filteredand concentrated to afford 0.110 g of the title compound as a whitesolid.

¹H NMR (DMSO-d₆, 400 MHz): δ 12.64 (br, 1H), 9.18 (s, 1H), 8.18 (d, 1H),7.17 (d, 1H), 7.12-7.06 (m, 4H), 7.01 (dt, 1H), 6.88-6.78 (m, 3H),6.66-6.62 (m, 3H), 6.02 (d, 1H), 5.85-5.60 (br, 2H), 5.42 (m, 1H),5.20-4.95 (br, 2H), 4.53-4.48 (m, 1H), 3.03 (dd, 1H), 2.88 (dd, 1H),2.10 (s, 3H), 2.08-2.04 (m, 4H), 1.67-1.54 (m, 4H).

Anal. Calcd. for C₃₆H₃₅N₃O₅+0.20 C₄H₁₀O: C, 71.53, H, 5.84, N, 6.95.Found: C, 71.28, H, 5.84, N, 6.76.

MS [(+)-ACPl, m/z]: 590.1 [M+H]⁺. Calcd. for C₃₆H₃₆N₃O₅: 590.2658.

EXAMPLE 11(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Example 5, Step E (0.300 g, 0.703 mmol), L-serine methyl esterhydrochloride (0.131 g, 0.844 mmol), 1-hydroxy benzotriazole (0.104 g,0.773 mmol) and 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.148 g, 0.773 mmol) were combined in amine-freeN,N-dimethylformamide (2.8 mL), followed by addition ofN,N-diisopropylethylamine (0.307 mL, 1.76 mmol). The reaction wasstirred at room temperature for 12 hours, then diluted with ethylacetate and washed with water, 1 N hydrochloric acid, 1 N sodiumhydroxide and brine. The combined aqueous washings were saturated withsodium chloride and extracted with ethyl acetate. The combined, organicphases were dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography onsilica gel eluting with 10% methanol in chloroform, to afford 0.330 g ofthe title compound as a pale yellow solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 8.10 (d, 1H), 7.30 (dd, 1H), 7.14-7.10 (m,2H), 7.03 (dt, 1H), 6.90-6.78 (m, 4H), 6.07 (d, 1H), 6.00-5.65 (br, 2H),5.42 (m, 1H), 5.25-4.95 (br, 3H), 4.52-4.47 (m, 1H), 3.76 (d, 2H), 3.66(s, 3H), 2.10 (s, 3H), 2.10-2.05 (m, 4H), 1.67-1.56 (m, 4H).

Anal. Calcd. for C₃₁H₃₃N₃O₅+0.10H₂O: C, 70.33, H, 6.32, N, 7.94. Found:C, 70.05, H 6.44, N, 7.66.

MS [(+)—ACPl, m/z]: 528.3 [M+H]⁺. Calcd. for C₃₁H₃₄N₃O₅: 528.2498.

EXAMPLE 12(2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester 0.25 hydrate

10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid (0.300 g, 0.703 mmol) of Example 5 Step E, D-serine methyl esterhydrochloride (0.131 g, 0.844 mmol), 1-hydroxy benzotriazole (0.104 g,0.773 mmol) and 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.148 g, 0.773 mmol) were combined in amine-freeN,N-dimethylformamide (2.8 mL), followed by the addition ofN,N-diisopropylethylamine (0.307 mL, 1.76 mmol). The reaction wasstirred at room temperature for 12 hours, then diluted with ethylacetate and washed with water, 1 N hydrochloric acid, 1 N sodiumhydroxide and brine. The combined aqueous washings were saturated withsodium chloride, and extracted with ethyl acetate. The organic phaseswere combined, dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography onsilica gel eluting with 10% methanol in chloroform, to afford 0.320 g ofthe title compound as a pale yellow solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 8.10 (d, 1H), 7.30 (dd, 1H), 7.14-7.10 (m,2H), 7.03 (dt, 1H), 6.90-6.78 (m, 4H), 6.07 (d, 1H), 6.00-5.65 (br, 2H),5.42 (m, 1H), 5.25-4.95 (br, 3H), 4.52-4.47 (m, 1H), 3.76 (d, 2H), 3.66(s, 3H), 2.10 (s, 3H), 2.10-2.05 (m, 4H), 1.67-1.56 (m, 4H).

Anal. Calcd. for C₃₁H₃₃N₃O₅+0.25H₂O: C, 69.97, H, 6.35, N, 7.90. Found:C, 69.63, H 6.25; N, 7.72.

MS [(+)—ACPl, m/z]: 528.3 [M+H]⁺. Calcd. for C₃₁H₃₃N₃O₅: 528.2498.

EXAMPLE 13(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid

(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester of Example 12 (0.260 g, 0.493 mmol), was dissolved inacetone (3.3 mL), followed by addition of 2.5 N sodium hydroxide (0.590mL, 1.48 mmol). The reaction was stirred at room temperature overnight,acidified with 1 N hydrochloric acid and extracted with diethyl ether.The combined ether phases were extracted with 1 N sodium hydroxide andthe basic extracts acidified with 2 N hydrochloric acid. The acidifiedextracts were extracted with diethyl ether and the combined extractsdried over anhydrous magnesium sulfate, filtered and concentrated toafford 0.210 g of the title compound as a white powder.

¹H NMR (DMSO-d₆, 400 MHz): δ 12.60 (br, 1H), 7.94 (d, 1H), 7.31 (dd,1H), 7.14 (s, 1H), 7.11 (dt, 1H), 7.03 (dt, 1H), 6.88 (dd, 1H),6.84-6.81 (m, 2H), 6.77 (d, 1H), 6.06 (d, 1H), 6.00-5.75 (br, 2H), 5.43(m, 1H), 5.25-4.90 (m, 3H), 4.46-4.41 (m, 1H), 3.76 (d, 2H), 2.11 (s,3H), 2.10-2.05 (m, 4H), 1.67-1.55 (m, 4H).

Anal. Calcd. for C₃₀H₃₁N₃O₅+0.50H₂O: C, 68.95, H, 6.17, N, 8.04. Found:C 68.77, H, 6.33, N, 8.01.

MS [(−)-ESI, m/z]: 512.4 [M−H]⁻. Calcd. for C₃₀H₃₀N₃O₅: 512.2192.

EXAMPLE 14(2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid

(2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester of Example 13 (0.260 g, 0.493 mmol), was dissolved inacetone (3.3 mL), followed by the addition of 2.5 N sodium hydroxide(0.590 mL, 1.48 mmol). The reaction was stirred at room temperatureovernight, acidified with 1 N hydrochloric acid and extracted withdiethyl ether. The combined ether phases were extracted with 1 N sodiumhydroxide and the basic extracts acidified with 2 N hydrochloric acid.The acidified extracts were extracted with diethyl ether and thecombined ether extracts dried over anhydrous magnesium sulfate, filteredand concentrated to afford 0.250 g of the title compound as a whitepowder.

¹H NMR (DMSO-d₆, 400 MHz): δ 12.60 (br, 1H), 7.94 (d, 1H), 7.31 (dd,1H), 7.14 (s, 1H), 7.11 (dt, 1H), 7.03 (dt, 1H), 6.88 (dd, 1H),6.84-6.81 (m, 2H), 6.77 (d, 1H), 6.06 (d, 1H), 6.00-5.75 (br, 2H), 5.43(m, 1H), 5.25-4.90 (m, 3H), 4.46-4.41 (m, 1H), 3.76 (d, 2H), 2.11 (s,3H), 2.10-2.05 (m, 4H), 1.67-1.55 (m, 4H),

Anal. Calcd. for C₃₀H₃₁N₃O₅+0.50H₂O: C, 68.95, H, 6.17, N, 8.04. Found:C, 68.93, H, 6.40, N, 7.92

MS [(−)-ESI, m/z]: 512.4 [M−H]⁻. Calcd. for C₃₀H₃₀N₃O_(5:)512.2192.

EXAMPLE 1510-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitolhemihydrate

Step A.(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[4-(3,4-dihydro-naphthalen-1-yl)-3-methyl-phenyl]-methanone

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[3-methyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-methanoneof Example 5, Step B (0.760 g, 1.89 mmol), 3,4-dihydro-naphthalen-1-yltrifluoromethanesulfonate (0.579 g, 2.08 mmol) anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (0.0460 g, 0.0567 mmol) were combined inN,N-dimethylformamide (9.5 mL). Aqueous sodium carbonate (2 M. 4.73 mL,9.45 mmol) was added and the was heated to 60° C. for 3 hours. Aftercooling to room temperature, the mixture was diluted with ethyl acetateand washed with water and brine. The organic phase was dried overanhydrous magnesium sulfate, filtered and concentrated and the residuepurified by flash column chromatography on silica gel eluting with 30%ethyl acetate in hexane. Recrystallization from petroleum ether afforded0.740 g of the title compound as white crystals, m.p.108° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.46 (d, 1H), 7.22-6.91 (m, 9H), 6.93 (s,1H), 6.22 (d, 1H), 5.95 (s, 1H), 5.92 (t, 1H), 5.84 (t, 1H), 5.30 (br,4H), 2.77 (t, 2H), 2.35-2.32 (m, 2H), 1.88 (s, 3H).

Anal. Calcd. for C₃₀H₂₆N₂O+0.16 C₆H₁₄+0.30H₂O: C, 82.68, H, 6.46, N,6.23. Found: C, 82.47, H, 6.63, N, 6.05.

MS [(+)-ACPl, m/z]: 431 [M+H]⁺. Calcd. for C₃₀H₂₇N₂O: 431.2128.

Step B.10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid

(10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-yl)-[4-(3,4-dihydro-naphthalen-1-yl)-3-methyl-phenyl]-methanoneof Step A (0.830 g, 1.93 mmol) and N,N-diisopropylethylamine (0.672 mL,3.86 mmol) were dissolved in anhydrous dichloromethane (9.7 mL).Trichloroacetyl chloride (0.646 mL, 5.79 mmol) was added dropwise andthe reaction stirred at room temperature overnight. The mixture wasdiluted with ethyl acetate and washed with aqueous sodium bicarbonate,1N hydrochloric acid and brine. The organic phase was dried overanhydrous magnesium sulfate, diluted with hexane and filtered throughsilica gel eluting with 30% ethyl acetate in hexane. The filtrate wasconcentrated to afford the crude trichloroacetate which was dissolved inacetone (12.9 mL) treated with 2.5 N sodium hydroxide (2.32 mL, 5.79mmol). The reaction was stirred at room temperature for 4 hours, thendiluted with 1 Nhydrochloric acid (75 mL) and extracted with diethylether. The combined organic phases were extracted with 1 N sodiumhydroxide and the combined basic extracts acidified with 2N hydrochloricacid, causing a precipitate to form. The precipitate was filtered,rinsed thoroughly with water and dried to afford 0.910 g of the titlecompound as a pale yellow powder, m.p. 150-153° C.

¹H NMR (DMSO-d₆, 400 MHz): δ 12.31 (br, 1H), 7.34 (dd, 1H), 7.19-7.00(m, 7H), 6.92 (d, 1H), 6.87 (d, 1H), 6.75 (d, 1H), 6.23 (d, 1H), 6.10(d, 1H), 5.95 (br, 2H), 5.85 (t, 1H), 5.19 (br, 2H), 2.78 (t, 2H), 2.34(br, 2H), 1.88 (s, 3H).

Anal. Calcd. for C₃₁H₂₆N₂O₃+1.50H₂O: C, 74.23, H, 5.83, N, 5.58. Found:C, 73.94, H, 5.45, N, 5.14.

MS [(+)—ACPl, m/z]: 475 [M+H]⁺. Calcd. for C₃₁H₂₇N₂O₃: 475.2018.

Step C.10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol

10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Step B (0.340 g, 0.716 mmol), N-methyl-D-glucamine (0.168 g,0.859 mmol), 1-hydroxy benzotriazole (0.106 g, 0.788 mmol) and1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.151 g,0.788 mmol) were combined in amine-free N,N-dimethylformamide (2.9 mL),followed by addition of N,N-diisopropylethylamine (0.186 mL, 1.07 mmol).The reaction was stirred at room temperature for 16 hours, then dilutedwith ethyl acetate and washed with water, 1 N hydrochloric acid, 1 Nsodium hydroxide and brine. The combined aqueous washings were saturatedwith sodium chloride and extracted with ethyl acetate. The combinedorganic phases were dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography onsilica gel eluting with a 10 to 15% gradient of methanol in chloroform,to afford 0.410 g of the title compound as a white solid.

¹H NMR (DMSO-d₆ +D₂O; 400 MHz): δ 7.38 (d, 1H), 7.17-6.96 (m, 8H),6.91-6.85 (m, 2H), 6.33 (br, 1H), 6.18 (d, 1H), 6.02 (br, 1H), 5.81 (t,1H), 5.31 (br, 2H), 3.90 (br, 1H), 3.57-3.31 (m, 6H), 3.04 (br, 3H),2.74 (t, 2H), 2.36-2.25 (m, 2H), 1.84 (s, 3H).

Anal. Calcd. for C₃₈H₄₁N₃O₇+0.50H₂O: C, 69.07, H, 6.41, N, 6.36. Found:C, 68.77, H, 6.54, N, 6.31.

MS [(+)—ACPl, m/z]: 652 [M+H]. Calcd. for C₃₈H₄₂N₃O₇: 652.3028.

EXAMPLE 16{10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}-3-carboxylicacid bis-(2-hydroxy-ethyl)-amide

10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Example 15, Step B (0.150 g, 0.316 mmol),bis(2-hydroxyethyl)amine (0.040 g, 0.379 mmol), 1-hydroxy benzotriazole(0.047 g, 0.348 mmol) and 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.067 g, 0.348 mmol) were combined in amine-freeN,N-dimethylformamide (1.3 mL), followed by addition ofN,N-diisopropylethylamine (0.083 mL, 0.474 mmol). The reaction wasstirred at room temperature for 16 hours, then diluted with ethylacetate and washed with 1 N hydrochloric acid, saturated aqueous sodiumbicarbonate, and brine. The combined aqueous washings were saturatedwith sodium chloride and extracted with ethyl acetate. The organicphases were combined, dried over anhydrous magnesium sulfate, filteredand concentrated. The residue was purified by flash columnchromatography on silica gel eluting with 7% methanol in chloroform toafford 0.160 g of the title compound as a white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.37 (dd, 1H), 7.19-7.00 (m, 7H), 6.92 (d,1H), 6.88 (d, 1H), 6.29 (d, 1H), 6.23 (d, 1H), 6.02 (d, 1H), 5.84 (t,1H), 5.32 (s, 2H), 5.31-4.98 (br, 2H), 4.81 (t, 2H), 3.55 (br, 8H), 2.78(t, 2H), 2.32 (br, 2H), 1.88 (s, 3H).

Anal. Calcd. for C₃₅H₃₅N₃O₄+0.50H₂O: C, 73.66, H, 6.36, N, 7.36. Found:C, 73.42, H 6.28, N, 7.41.

MS [(+)-ESI, m/z]: 562 [M+H]⁺, 579 [M+Na]⁺. Calcd. for C₃₅H₃₆N₃O₄:562.2708.

EXAMPLE 17{10-[4-(1-Cyclohex-1-en-1-yl)-3-methylbenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}[4-(2-hydroxyethyl)-1-piperazinyl]methanone

Prepared by acylation of10-(4-cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid of Example 5, Step E with 1-(2-hydroxyethyl)-piperazine in themanner of Example 5, Step H.

MS [ESI(+), m/z]: 539.96 [M+H]⁺. Calcd. for C₃₃H₃₉N₄O₃ 539.3022.

1. A compound of the formula (I):

R₁ and R₂ are, independently, selected from hydrogen, (C₁-C₆ )loweralkyl, halogen, cyano, trifluoromethyl, hydroxy, amino, (C₁-C₆) loweralkylamino, (C₁-C₆) lower alkoxy, —OCF₃, (C₁-C₆) lower alkoxycarbonyl,—NHCO[(C₁-C₆ )lower alkyl], carboxy, —CONH₂, —CONH(C₁-C₆) lower alkyl,or —CON[(C₁-C₆) lower alkyl]₂; R₃ is a substituent selected fromhydrogen, (C₁-C₆ ) lower alkyl, (C₁-C₆) lower alkoxy, hydroxy, amino,(C₁-C₆) lower alkylamino, —CO lower alkyl (C₁-C₆), or halogen; R₄consists of the moiety B—C; wherein B is selected from the group of

and C is defined as:

wherein: A is CH or N; R₅, R₆, R₇ and R₈ are independently, selectedfrom hydrogen, (C₁-C₆) lower alkyl, (C₁-C₆) lower alkoxy, (C₁-C₆) loweralkylcarbonyl, (C₃-C₆) lower alkenyl, (C₃-C₆) lower alkynyl, (C₁-C₆)lower alkyl, hydroxy (C₁-C₆) lower alkyl, alkoxy (C₁-C₆) lower alkyl,acyloxy (C₁-C₆), (C₃-C₈) cycloalkyl, formyl, cycloalkylcarbonyl,carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, aryl alkoxycarbonyl,carbamoyl, —O—CH₂—CN═CH₂, halogen, halo lower alkyl, trifluoromethyl,—OCF₃, —S(lower alkyl), —OC(O)N[lower alkyl]₂, —CONH(lower alkyl),—CON[lower alkyl]₂, lower alkylamino, di-lower alkylamino, lower alkyldi-lower alkylamino, hydroxy, cyano, trifluoromethylthio, nitro, amino,lower alkylsulfonyl, aminosulfonyl, lower alkylaminosulfonyl, phenyl,naphthyl, or

R₉ is chosen from the group of hydrogen, (C₁-C₆) lower alkyl,alkoxycarbonyl, —CON[(C₁-C₆) lower alkyl]₂, cyano; or aryl, optionallysubstituted by halogen, or (C₁-C₆) lower alkoxy; R₁₀ represents one totwo substituents chosen independently, from the group of hydrogen,(C₁-C₆) lower alkyl, [(C₁-C₆) lower alkyl]₂, carbonyl,

 azido, amino, —NH[lower alkyl], —N[lower alkyl]₂, amino carbonyl loweralkyl, phthalimido, cyano, halogen, thio lower alkyl, aryloxy, arylthio,aryl optionally substituted with one to three substituents chosen from(C₁-C₆) lower alkyl, alkoxy or halogen; hydroxy, lower alkoxy, —OSO₂R₃₂,or OP′ wherein P′ is tert-butyl dimethylsilyl, tert-butyl diphenylsilyl,carbonyl loweralkyl, carbonyl trifluoro lower alkyl, aryl lower alkyl,arylcarbonyl, methoxymethyl, or methylthiomethyl; with the proviso thatwhen R₁₀ represents two substituents, the two substituents may be joinedtogether to form with the cyclohexene ring to which they are attachedbicyclo[3.2.1]oct-2-ene, or (6,6-dimethyl)-bicyclo[3.1.1]hept-2-ene; andR is selected from any of the following groups:

wherein: R₁₁ is selected from the group of hydrogen, (C₁-C₆) loweralkyl, (C₇-C₁₂) aryl lower alkyl, wherein the aryl moiety is optionallysubstituted with lower alkoxy, or from the group of:

R₁₂ is selected from any of the group of:

R₁₃ and R₁₄ are independently, chosen from the group of hydrogen,(C₁-C₆) lower alkyl or (C₇-C₁₂) aryl lower alkyl; R₁₅ is hydrogen, or(C₁-C₆) lower alkyl; R₁₆ is hydroxy, (C₁-C₆) lower alkoxy, or OP whereinP is a hydroxy protecting group; R₁₇ is selected from the group ofhydrogen, (C₁-C₆) lower alkyl or (C₇-C₁₂) aryl lower alkyl; R₁₈ isselected from the group of —N[lower alkyl]₂, or —N[aryl lower alkyl]₂;

R₁₉ is hydrogen, or (C₁-C₆) lower alkyl, or R₂₇; R₂₀ is selected fromthe group of (C₁-C₆) lower alkyl, —COR₁₆, —CONH[lower alkyl], —CON[loweralkyl]₂; R₂₁ is aryl, optionally substituted by one to threesubstituents chosen from hydroxy, (C₁-C₆) lower alkoxy, aryloxy loweralkyl, or halogen; R₂₂ represents one to four substituents chosen,independently, from the group of hydrogen or (C₁-C₆) lower alkyl; R₂₃ isselected from the group of

R₂₄ is (C₁-C₆) lower alkyl, or aryl (C₁-C₆) lower alkyl; R₂₅ and R₂₆taken together represent one to four substituents chosen, independently,from the group of R₁₆, R₂₇, (C₁-C₆) lower alkyl, [(C₁-C₆) lower alkyl]₂,—CONH[lower alkyl], —CON[lower alkyl]₂, R₃₀,

 with the proviso that at least one of said one to four substituents isnot (C₁-C₆) lower alkyl, —[(C₁-C₆) lower alkyl]₂, —CONH[(C₁-C₆) loweralkyl] or —CON[C₁-C₆) lower alkyl]₂; R₂₇ and R₂₈ are selected from thegroup of hydroxy (C₁-C₆) lower alkyl, lower alkoxy (C₁-C₆) lower alkyl,or (C₁-C₆) lower alkyl OP, wherein P is a hydroxy protecting group; R₂₉represents one to four substituents chosen, independently, from thegroup of R₁₆ or R₂₇; R₃₀ is

R₃₁ is hydrogen, or (C₁-C₆) lower alkyl; X and Y are either CH, or N; pis an integer from 0 to 1; q is an integer from 2 to 4; r is an integerfrom 0 to 3; s is an integer from 0 to 2; and t is an integer from 1 to2; or the pharmaceutically acceptable salts thereof.
 2. A compound ofthe formula:

R₁ and R₂ are, independently, selected from hydrogen, (C₁-C₆ )loweralkyl, halogen, cyano, trifluoromethyl, hydroxy, amino, (C₁-C₆) loweralkylamino, (C₁-C₆) lower alkoxy, —OCF₃, (C₁-C₆) lower alkoxycarbonyl,—NHCO[(C₁-C₆ )lower alkyl], carboxy, —CONH₂, —CONH (C₁-C₆) lower alkyl,or —CON[(C₁-C₆) lower alkyl]₂; R₃ is a substituent selected fromhydrogen, (C₁-C₆) lower alkyl, (C₁-C₆) lower alkoxy, hydroxy, amino,(C₁-C₆) lower alkylamino, —CO lower alkyl (C₁-C₆), or halogen; R₄consists of the moiety B—C; wherein B is selected from the group of

and C is defined as:

wherein: A is CH or N; R₅, R₆, R₇ and R₈ are independently selected fromhydrogen, (C₁-C₆) lower alkyl, (C₁-C₆) lower alkoxy, (C₁-C₆) loweralkylcarbonyl, (C₃-C₆) lower alkenyl, (C₃-C₆) lower alkynyl, (C₁-C₆)lower alkyl, hydroxy (C₁-C₆) lower alkyl, alkoxy (C₁-C₆) lower alkyl,acyloxy (C₁-C₆), (C₃-C₈) cycloalkyl, formyl, cycloalkylcarbonyl,carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, aryl alkoxycarbonyl,carbamoyl, —O—CH₂—CH═CH₂, halogen, halo lower alkyl, trifluoromethyl,—OCF₃, —S(lower alkyl), —OC(O)N[lower alkyl]₂, —CONH(lower alkyl),—CON[lower alkyl]₂, lower alkylamino, di-lower alkylamino, lower alkyldi-lower alkylamino, hydroxy, cyano, trifluoromethylthio, nitro, amino,lower alkylsulfonyl, aminosulfonyl, or lower alkylaminosulfonyl; R₉ ischosen from the group of hydrogen, (C₁-C₆)lower alkyl, alkoxycarbonyl,—CON[(C₁-C₆) lower alkyl]₂, or cyano; R₁₀ represents one to twosubstituents chosen independently, from the group of hydrogen, (C₁-C₆)lower alkyl, [(C₁-C₆) lower alkyl]₂, carbonyl, azido, amino, —NH[loweralkyl], —N[lower alkyl]₂, amino carbonyl lower alkyl, phthalimido,cyano, halogen, thio lower alkyl, aryloxy, arylthio, hydroxy, loweralkoxy, —OSO₂R₃₂, or OP′ wherein P′ is tert-butyl dimethylsilyl,tert-butyl diphenylsilyl, carbonyl loweralkyl, carbonyl trifluoro loweralkyl, aryl lower alkyl, arylcarbonyl, methoxymethyl, ormethylthiomethyl; and R is selected from any of the following groups:

wherein: R₁₁ is selected from the group of hydrogen, (C₁-C₆) loweralkyl, (C₇-C₁₂) aryl lower alkyl, wherein the aryl moiety is optionallysubstituted with lower alkoxy, or any of the following groups:

R₁₂ is selected from any of the following groups:

R₁₃ and R₁₄ are independently, chosen from the group of hydrogen,(C₁-C₆), lower alkyl or (C₇-C₁₂) aryl lower alkyl; R₁₆ is hydroxy,(C₁-C₆) lower alkoxy, or OP wherein P is a hydroxy protecting group; R₁₇is selected from the group of hydrogen, (C₁-C₆) lower alkyl or (C₇-C₁₂)aryl lower alkyl; R₁₈ is selected from the group of —N[(C₁-C₆) loweralkyl]₂,

R₁₉ is hydrogen, or R₂₇; R₂₀ is selected from the group of —COR₁₆,—CONH[(C₁-C₆) lower alkyl], —CON[(C₁-C₆) lower alkyl]₂; R₂₁ is aryl,optionally substituted by one to three substituents chosen from hydroxy,or (C₁-C₆) lower alkoxy; R₂₃ is selected from the group of

R₂₄ is (C₁-C₆) lower alkyl, or aryl (C₁-C₆) lower alkyl; R₂₅ and R₂₆taken together represent one to four substituents chosen, independently,from the group of R₁₆, R₂₇, (C₁-C₆) lower alkyl, [(C₁-C₆) lower alkyl]₂,—CONH[(C₁-C₆) lower alkyl], —CON[(C₁-C₆) lower alkyl]₂, R₃₀,

 with the proviso that at least one of said one to four substituents isnot (C₁-C₆) lower alkyl, —[(C₁-C₆) lower alkyl]₂, —CONH[lower alkyl] or—CON[lower alkyl]₂; R₂₇ and R₂₈ are selected from the group of hydroxy(C₁-C₆) lower alkyl, lower alkoxy (C₁-C₆) lower alkyl, or (C₁-C₆) loweralkyl OP, wherein P is a hydroxy protecting group; R₂₉ represents one tofour substituents chosen, independently, from the group of R₁₆ or R₂₇;R₃₀ is

R₃₁ is hydrogen, or (C₁-C₆) lower alkyl; R₃₂ is selected from the groupof (C₁-C₆) lower alkyl, trifluoro lower alkyl, or aryl optionallysubstituted by lower alkyl; X and Y are either CH, or N; p is an integerfrom 0 to 1; q is an integer from 2 to 4; r is an integer from 0 to 3; sis an integer from 0 to 2; and t is an integer from 1 to 2; or thepharmaceutically acceptable salts thereof.
 3. A compound of claim 1selected from the group of:1-[[[10-[5-Chloro-4-(1-cyclohex-1-en-1-yl)-2-methoxybenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol;10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;or10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-N-(2,3-dihydroxypropyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;or a pharmaceutically acceptable salt form thereof.
 4. A compound ofclaim 1 selected from the group of:10-[4-(Cyclohex-1-en-1-yl)-3-methyl-benzoyl]-10,11-dohydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-deoxy-D-glucitol;10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-((2S)-2-{(4R,5R)-5-[(1R)-1,2-dihydroxyethyl]-2-oxo-1,3-dioxolan-4-yl}-2-hydroxyethyl)-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;or10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-[((4S,5S)-5-{(R)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-2-oxo-1,3-dioxolan-4-yl)methyl]-N-methyl-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;or a pharmaceutically acceptable salt form thereof.
 5. A compound ofclaim 1 selected from the group of:10-(4-Cyclohex-1-en-1-yl-3-methylbenzoyl)-N-methyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentamethoxyhexyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxamide;(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionicacid ethyl ester;(2S)-2-{[10-(4-Cyclohex-1-en-1-yl)-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]-amino}-3-(4-hydroxy-phenyl)-propionicacid;(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester; or(2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid methyl ester; or a pharmaceutically acceptable salt form thereof.6. A compound of claim 1 selected from the group of:(2S)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid;(2R)-2-{[10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carbonyl]-amino}-3-hydroxy-propionicacid;10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]carbonyl]methylamino]-1-depxy-D-glucitol;{10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}-3-carboxyticacid bis-(2-hydroxy-ethyl)-amide; or{10-[4-(1-Cyclohexen-1-yl)-3-methylbenzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl}[4-(2-hydroxyethyl)-1-piperazinyl]methanone;or a pharmaceutically acceptable salt form thereof.
 7. A methodcomprising contacting an oxytocin receptor with a compound of claim 1.8. A method of treating disorders which are remedied or alleviated byoxytocin antagonist activity in a mammal, the method comprisingadministering to the mammal in need thereof a pharmaceutically effectiveamount of a compound of claim 1, wherein the disorder which is remediedor alleviated by oxytocin antagonist activity is dysmenorrhea.
 9. Amethod of treating disorders which are remedied or alleviated byoxytocin antagonist activity in a mammal, the method comprisingadministering to the mammal in need thereof a pharmaceutically effectiveamount of a compound of claim 1, wherein the disorder which is remediedor alleviated by oxytocin antagonist activity is endometritis.
 10. Amethod for treating or preventing preterm labor in a mammal, the methodcomprising administering to the mammal in need thereof apharmaceutically effective amount of a compound of claim
 1. 11. A methodfor suppressing labor prior to caesarean delivery in a mammal, themethod comprising administering to the mammal in need thereof apharmaceutically effective amount of a compound of claim
 1. 12. Apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of claim 1, and a pharmaceutically acceptablecarrier or excipient.
 13. A compound selected from:10-(5-Chloro-4-cyclohex-1-en-1-yl-2-methoxybenzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid;2,2,2-Trichloro-1-[10-(4-cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-3-yl]-ethanone10-(4-Cyclohex-1-en-1-yl-3-methyl-benzoyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid;10-[4-(3,4-Dihydro-naphthalen-1-yl)-3-methyl-benzoyl]-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-3-carboxylicacid; or a pharmaceutically acceptable salt form thereof.
 14. A methodfor suppressing labor prior to caesarean delivery in a mammal, themethod comprising administering to the mammal in need thereof apharmaceutically effective amount of a compound of claim
 13. 15. Apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of claim 13 and a pharmaceutically acceptablecarrier or excipient.
 16. A method of antagonizing the oxytocin receptorin a mammal comprising administering to the mammal a pharmaceuticallyeffective amount of a compound of claim
 1. 17. A method of treatingdisorders which are remedied or alleviated by oxytocin antagonistactivity in a mammal, the method comprising administering to the mammalin need thereof a pharmaceutically effective amount of a compound ofclaim 13, wherein the disorder which is remedied or alleviated byoxytocin antagonist activity is dysmenorrhea.
 18. A method of treatingdisorders which are remedied or alleviated by oxytocin antagonistactivity in a mammal, the method comprising administering to the mammalin need thereof a pharmaceutically effective amount of a compound ofclaim 13, wherein the disorder which is remedied or alleviated byoxytocin antagonist activity is endometritis.
 19. A method for treatingor preventing preterm labor in a mammal, the method comprisingadministering to the mammal in need thereof a pharmaceutically effectiveamount of a compound of claim 13.